EXPLORINGTHEUTILIZATIONOF CRITHIDIAFASCICULATA ASAMODELORGANISMTOSTUDYPATHOGENIC KINETOPLASMIDS

Wakisa Kipandula

A Thesis Submitted for the Degree of MPhil at the University of St Andrews

2017

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Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/12217

This item is protected by original copyright Exploring the utilization of fasciculata as a model organism to study pathogenic kinetoplastids

Wakisa Kipandula

This thesis is submitted in partial fulfilment for the degree of MPhil at the University of St Andrews

Date of Submission May 2017

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Abstract

This study aimed to explore the utilization of C. fasciculata as a convenient model organism to study the cell biology and drug discovery vehicle of the pathogenic kinetoplastids. We specifically aimed to: (i) develop and validate aprotein A-TEV-protein C (PTP) tagged protein expression system for C. fasciculata, (ii) develop a Resazurin-reduction viability assay with C. fasciculata and use this for subsequent screening for anti-crithidial compounds from the GSK open access pathogen boxes, and (iii) to study the effects of ionizing gamma radiation on C. fasciculata.

We report the construction of plasmid pNUS-PTPcH, which can be utilised to express PTP tagged kinetoplastids proteins in C. fasciculata for subsequent purification. As a proof of concept, we have shown that C. fasciculata can be efficiently transfected with this plasmid and facilitate the isolation of two protein complexes: replication factor C (RFC) and the exosome. We have demonstrated that the expressed PTP tagged-replication factor C subunit 3 (PTP-RFC3) co-purifies with RFC1, RFC2, RFC4, RFC5 and RAD17, and that the PTP tagged exosome subunit RRP4 co-purifies with RRP6, EAP1, RRP45, RRP40, RRP41B, CSL4, EAP2, RRP41A and EAP4. In addition, this thesis reports the development of a resazurin-reduction cell viability assay in C. fasciculata and reveals attractive core chemical scaffolds present in more than one of the open access GSK pathogen boxes, which will be followed up against the actual pathogenic kinetoplastids. Furthermore, this study has demonstrated that compared to cultured forms of T. cruzi which undergo growth arrest for 96 hours after exposure to 500 Gy of gamma radiation, C. fasciculata is able to recover and resume normal growth within 24 hours after being subjected to doses as high as 1000Gy.

The constructed plasmid, the identified chemical scaffolds and the observed responses of C. fasciculata to gamma irradiation will help facilitate further studies aimed to discover novel drugs for kinetoplastid diseases.

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Declaration

I, Wakisa Kipandula, hereby certify that this thesis, which is approximately 30,000 words in length, has been written by me, and that it is the record of work carried out by me, with the exception of a single plasmid consruction carried out by Dr Stuart MacNeill, and that it has not been submitted in any previous application for a higher degree.

I was admitted as an MPhil student in November 2014. The work was carried out in University of St Andrews and the University Of Malawi College Of Medicine between 2014 and 2016.

Date: 10th November 2017 Signature of candidate:

We hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of MPhil in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree.

Date Signatures of supervisors:

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In submitting this thesis to the University of St Andrews, I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any third-party copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo below.

The following is an agreed request by candidate and supervisor regarding the electronic publication of this thesis:

(i) Access to printed copy and electronic publication of thesis through the University of St Andrews.

Date: 10th November 2017 Signature of candidate:

Date Signatures of supervisors:

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Acknowledgements

I thank my supervisors Dr Stuart MacNeill and Professor Terry Smith for their valuable guidance, support, advice and motivation while working on this highly interesting project. I acknowledge the assistance offered to me by staff and fellow students in the Biomedical Sciences Research Complex, level 3.

Special thanks to Wilberforce Sabiiti, Beatrice Mwagomba, Kondwani Katundu, Daniel Khomba and everybody who made my stay in very cold and boring St Andrews worthwhile. You are such a wonderful people.

I am indebted to E. Tetaud for providing C. fasciculata pNus plasmids. This work would not have been possible without generous funding through the Global Health Implementation Programme at the University of St Andrews by the Gloag Foundation and the Western Union Foundation.

It is with immense gratitude that I acknowledge the management of the University Of Malawi College Of Medicine for granting me a study leave. I am grateful to my wife Wezzie for her encouragement and perseverance while I was away. You are such a strong woman.

Last but not least, the one above all of us, the omnipresent God for answering my prayers and for giving me the strength to plod on despite my constitution wanting to give up this work. Thank you so much Dear Lord.

This work is dedicated to my son Andile.

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Publications Some of the results from this thesis have been published in a well-recognised scientific journal as follows. i. Kipandula W, Smith TK, MacNeill SA. Tandem affinity purification of exosome and replication factor C complexes from the non-human infectious kinetoplastid parasite Crithidia fasciculata. Molecular and Biochemical Parasitology 217: 19-22 (2017). Available from, DOI: 10.1016/j.molbiopara.2017.08.004

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Table of contents

Abstract ...... ii Declaration ...... iii Acknowledgements ...... v Publications…………………………………………………………………………………………………………………………….……….vi

List of Abbreviations ...... x 1. Chapter 1: Background introduction ...... 1 1.1 Introduction ...... 1 1.2 Current treatment for disease caused by kinetoplastids ...... 4 1.3 Challenges to research on pathogenic kinetoplastids ...... 6 1.4 C. fasciculata as a model organism to study kinetoplastid biology ...... 7 1.5 Aims of the study ...... 9 2. Chapter 2: Developing and validating an expression vector for subsequent isolation of PTP tagged kinetoplastids proteins in C. fasciculata ...... 10 2.1 Introduction ...... 10 2.1.1 Kinetoplastids proteins expression systems ...... 10 2.1.2 Multi protein complexes ...... 11 2.1.3 Overview of protein complexes purification methods ...... 17 2.2 Materials and methods ...... 21 2.2.1 Organisms and reagents ...... 21 2.2.2 General Molecular biology techniques ...... 21 2.2.3 Construction of the expression vector (pNUS-PTPcH) ...... 23 2.2.4 Identification of C. fasciculata and T. brucei homologous proteins from yeast RFC and exosome complexes ...... 23 2.2.5 Cloning of the target subunits ...... 24 2.2.6 Parasite transfection and generation of cell lines ...... 24 2.2.7 Expression and PTP purification of the proteins ...... 24 2.2.8 Mass Spectroscopy analysis ...... 26 2.3 Results and Discussion ...... 26 2.3.1 Identification of C. fasciculata and T. brucei homologous proteins from yeast RFC and exosome complexes ...... 26 2.3.2 Construction of the expression vector ...... 27 2.3.3 Cloning of C. fasciculata RFC3 and RRP4 subunits in pNUS-PTPcH plasmid and Transfection...... 29

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2.3.4 Expression and PTP purification of the proteins ...... 30 3. Chapter 3: Developing a Resazurin-viability assay in Crithidia fasciculata allowing subsequent screening for Anti-Crithidial Compounds from the GSK Open Access Pathogen Boxes ...... 35 3.1 Introduction ...... 35 3.1.1 Open access chemical boxes ...... 36 3.1.2 High throughput phenotypic screening assays ...... 37 3.1.3 Resazurin-reduction cell-based HTS assay ...... 38 3.2 Materials and methods ...... 40 3.2.1 Parasites and cell culture ...... 40 3.2.2 Compounds libraries ...... 40 3.2.3 Resazurin-reduction C. fasciculata cell-based assay optimization ...... 41 3.2.4 Compound sensitivity assays ...... 42 3.3 Results and Discussion ...... 44 3.3.1 Resazurin-reduction C. fasciculata cell-based assay optimization ...... 44 3.3.2 Screening GSK pathogen boxes for anti-crithidial compounds with a resazurin- reduction assay...... 49 4. Chapter 4: The effects of ionizing gamma radiation on the kinetoplastid Crithidia fasciculata ...... 59 4.1 Introduction ...... 59 4.1.1 Gamma ionizing irradiation and its effects on cells ...... 59 4.1.2 Effects of gamma irradiation stress on Kinetoplastids ...... 62 4.2 Experimental procedures ...... 63 4.2.1 Parasites strain and cell culture ...... 63 4.2.2 Irradiation of parasites ...... 63 4.2.3 Growth experiments ...... 63 4.2.4 Parasites Viability experiments ...... 64 4.2.5 Parasites motility estimates ...... 64 4.2.6 Parasites morphology ...... 64 4.3 Results and Discussion ...... 65 5. Chapter 5: General discussion and Principle conclusions ...... 71 5.1 General Discussion ...... 71 5.2 Principle conclusions ...... Error! Bookmark not defined. 6. References ...... 80 7. Appendices ...... 92 7.1 Appendix 1. The C. fasciculata serum free defined growth media recipe...... 92 7.2 Appendix 2. Extraction of C. fasciculata genomic DNA...... 92

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7.3 Appendix 3. The designed forward and reverse primers that were used to amplify the ORFs of target subunits...... 93 7.4 Appendix 4(a). A conventional PCR reaction recipe and cycling conditions using Q5 High- fidelity DNA polymerase enzyme...... 93 7.5 Appendix 4(b). PCR reaction recipe and cycling conditions for a standard My Taq Red MIX. 94 7.6 Appendix 5. Preparative restriction/diagnostic digest and ligation experimental set up. ... 94 7.7 Appendix 6. Recipes for solution and buffers used for PTP purification experiments ...... 95 7.8 Appendix 7(a). Protein sequences of RFC complex subunits identified by Mass spectroscopic analysis...... 96 7.9 Appendix 7(b). Protein sequences of exosome complex subunits identified by Mass spectroscopic analysis...... 98 7.10 Appendix 8. The full sequence of the constructed pNUS-PTPcH plasmid ...... 101 7.11 Appendix 9. Profiles and percentage anticrithidial activities of compounds in the MMV pathogen box ...... 102 7.12 Appendix 10. Profiles and percentage anticrithidial activities of compounds in the GSK T.blucei box...... 112 7.13 Appendix 11. Profiles and percentage anticrithidial activities of compounds in the GSK T. cruzi box...... 116 7.14 Appendix 12. Profiles and percentage anticrithidial activities of compounds in the GSK box ...... 121

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List of Abbreviations

°C Degrees Celsius 3’ 3 prime DNA end 5’ 5 prime DNA end AAA+ ATPase associated with a variety of cellular activities ATP Adenosine triphosphate bp Base pairs CBP Calmodulin binding protein CL Cutaneous CNS Central nervous system Csl4 Cep1 Synthetic Lethal Ctf8 Chromosome transmission fidelity protein 8 Cyp51 Cytochrome 51 Da Dalton DCC1 DNA replication and sister chromatid cohesion 1 DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid

EC50 Effective concentration inhibiting 50% cell proliferation EDTA Ethylenediamine tetra-acetic acid EGTA Ethylene glycol-bis (2-aminoethylether)-N,N,N',N'- tetraacetic acid Elg1 Enhanced level of genomic instability 1 FDA Food and drug administration g Gram GE Gut epithelium GSK Glaxo Smith Kline GSPS Glutathionylspermidine synthetase Gys Grays HAT Human Hr Hour HTS High throughput screening ID Identity IFN Interfelon IgG Immunoglobulin G IG-PGKAB Intergenic-phosphoglycerate kinase genes A and B IL-2 Interleukin-2 IR Ionising radiation KAP1 Kruppel-Associated protein 1 kb Kilobase kDa Kilodalton kDNA DNA l Litre

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LB Luria Bertani medium M Molar MALDI Matrix-Assisted Laser Desorption/Ionisation mg Milligram ml Millilitre ML Mucosal leishmaniasis mM Millimolar MMV Medicines for Malaria Venture MRC-5 Medical Research Council cell strain 5 mtDNA Mitochondrial DNA Mw Molecular weight NAD Nicotinamide adenine NADPH Nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnogy Information nDNA Nuclear DNA NECT - combination therapy nm Nanometre NTD Neglected tropical disease ORF Open reading frame PAP Peroxidase Anti-peroxodase PBS Phosphate buffered saline PBS-T PBS-Tween PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PDPs product development partnerships pIC50 - log of 50% inhibitory concentration Pol Polymerase PPP Public-private partnerships PTP Protein A-TEV-Protein C RFC Replication factor C RLC RFC-like complexes RNS Reactive nitrogen species ROS Reactive oxygen species rpm Revolutions per minute rRNA ribosomal RNA RRP4 Ribosomal RNA processing 4 SAR Structure activity relationship SB Sample buffer for protein gel electrophoresis SDS Sodium dodecyl sulfate SE Standard error Sno RNA Small nucleolar RNA TAP Tandem affinity purification TE Tris EDTA TEV Tobacco etch virus

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Tris 2-Amino-2-hydroxymethyl-propane-1, 3-diol Tris (hydroxymethyl) aminomethane tRNA Transfer RNA TSB Transformation storage buffer UTR Untranslated regions UV Ultraviolet V Volt VL Visceral leishmaniasis VSG Variant surface glycoprotein wt Wild-type µg Microgram µl Microlitre µm Micrometre

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1. Chapter 1: Background introduction

1.1 Introduction Kinetoplastids comprise of a group of flagellated protozoans that cause fatal diseases in humans and other mammals. They are characterised by the presence of a DNA-containing region, known as a “kinetoplast,” in their single large mitochondrion (Stuart et al., 2008). Common human diseases that are caused by these kinetoplastids include; human African trypanosomiasis (HAT), also known as African sleeping sickness, which is caused by two of the three subspecies of (rhodesience and gambiense), human American trypanosomiasis also known as , which is caused by Trypanosoma cruzi; and various forms of leishmaniasis which are caused by different species of Leishmania (Burri and Brun, 2003). T. brucei, T. cruzi and Leishmania species are together further grouped into the Trypanosomatidae, commonly referred to as the ‘TriTryps’. It is estimated that about a half a billion-people living in tropical and subtropical areas of the world are at risk of contracting the diseases caused by the TriTryps, with more than 20 million individuals infected with the pathogens that cause them resulting in extensive suffering and more than 100,000 deaths per year (Stuart et al., 2008). The cellular biology of the various kinetoplastids is very similar. For example, they are all motile protozoans with a single that originates near their large single mitochondrion and emanates from their flagellar pocket in the cell membrane, the only place where endo- and exocytosis also takes place. Their peroxisomes are modified to perform glycolysis, and thus referred to as glycosomes. Their plasma membrane is underlain with a corset of microtubules and their cell surface is highly decorated with species-specific molecules that are critical for their survival. They typically grow asexually although sexual recombination has been observed in T. brucei, T. cruzi, and in some Leishmania species. Nevertheless, they divide by binary fission, during which their nucleus does not undergo membrane dissolution or chromosome condensation (Stuart et al., 2008). Kinetoplastids have a unique genome organisation, which consists of unidirectional large gene clusters, which are transcribed polycistronically (Teixeira et al., 2011). They possess distinct genetic processes including RNA polymerase I-mediated transcription and trans-splicing of the immature RNA, which is followed by the addition of a 5’ mini-exon and 5’-polyadenylation to form mature RNA transcripts (Hajduk and Ochsenreiter, 2010). Although the kinetoplastids share such many similarities, they cause very distinctive forms of disease as shown in Table 1.

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Sleeping sickness Chagas disease Leishmaniasis Stages Early (hemolymphatic) Acute phase, VL, CL stage, late (CNS) stage indeterminant phase, chronic phase (cardiac and digestive forms) Causative T.b. gambiense, T.b. T. cruzi ∼21 Leishmania agent rhodesiense spp. e.g., L. donovani (VL), L. braziliensis, L. major (CL) Host cell Extracellular, in blood, Intracellular, in Intracellular, in lymph, cerebral spinal fluid, cytoplasm of heart, phagolysosomes of and intercellular spaces smooth muscle, gut, macrophages CNS, and adipose tissue cells Vectors Tsetse flies (∼20 Glossina Reduviid bugs Phebotomine spp.) (palpalis group, T.b. (∼12/∼138 sandflies (∼70 gambiense; morsitans Triatominae spp.) Phlebotomus spp. in group, T.b. rhodesiense) (Triatoma, Old World, Rhodnius, and Lutzomyia spp. in Panstrongylus spp.) New World) Mode of Infected fly bite, congenital Contamination by Infected fly bite transmission (rare), blood transfusion feces of infected (rare) bugs (e.g., at bite site, in mucous membranes, in food or drink),blood transfusion, congenital, organ transplantation (rare) Geographic Sub-Saharan Africa (∼20 South and Central South and Central distribution countries) America (19 America, Europe, countries) Africa, Asia (88 endemic countries) Population 50 million 100 million 350 million at risk Infected 70,000–80,000 8–11 million 12 million Deaths/year ∼30,000 14,000 51,000 (VL)

Table 1. Details of some kinetoplastid diseases. The data was copied from Maudlin et al. (2004) and Lane et al. (1993). Non-standard abbreviations: CL (Cutaneous leishmaniasis), ML (Mucosal leishmaniasis) and VL (Visceral leishmaniasis).

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The general life cycles of kinetoplastids vary according to the invertebrate vector that is involved in the transmission. As described by Bartholomeu et al. (2014), Leishmania species multiply as promastigotes in the sand fly’s mid-gut. These parasites get injected into the human host as the fly is taking a blood meal and consequently get engulfed by macrophages. Inside the microphages, the parasites develop and proliferate as amastigotes and infect other surrounding cells after cell lysis (Fig 1a).

Trypanosoma cruzi parasites proliferate as epimastigotes in the revuviid bug’s mid-gut and spread to colonise the bug’s intestines. In the intestines, the parasite develops into infective metacyclic promastigotes forms get excreted alongside the bug’s faeces (Fig. 1b). T. cruzi parasites target any host cell with a nuclear through recruitment of lysosomal or simply invagination the cell’s plasma membrane (Bartholomeu et al., 2014). They then proliferate into amastigotes and trypomastigotes in the cytosol and realised by lysis of infected cells, which are taken up by the surrounding cells (Ueno and Wilson, 2012).

Unlike T. cruzi and most Leishmania species, T. brucei parasites proliferate outside the host cells for their entire life cycle (Bartholomeu et al., 2014) (Fig. 1c). The parasites proliferate in tsetse fly intestines as procyclic forms, which develop into infective metacyclic forms in the salivary glands. The metacyclic parasites are injected into the host through saliva when the fly is taking a blood meal and multiply in the host blood as procyclic trypomastigotes. However, since the T.brucei parasites cannot invade and hide inside the host cells like T. cruzi and L.major, the parasite had to adapt to a mechanism, which avoids being attacked by the host immune response. This is achieved by the parasite’s cell-surface “Variant surface glycoprotein” (VSG) which undergoes antigenic variation and avoid the parasite’s recognition by the host immune system (Horn and McCulloch, 2010). Through this mechanism, the parasites are always one-step ahead of the host immune responses.

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Figure 1. Life cycles of the kinetoplastids. (a) L. major multiplies as promastigotes in the mid-gut of a sand fly and are transmitted to the host as metacyclic promastigotes during a blood meal. In the host, the parasites are phagocytosed by macrophages and the metacyclic forms are converted into amastigotes, which multiply numerous times before being released during cell lysis. (b) T. cruzi epimastigotes in the mid-gut of a bug transform into infective metacyclic trypomastigotes in the bug’s rectum and get excreted alongside the bud’s faeces to invade the host’s nucleated cells. (c) T. brucei multiplies as procyclic forms in the intestinal tract of tsetse fly and transformed into infective metacyclic forms in the fly’s salivary glands. When taking the blood, the fly injects the parasites into the host blood, which then proliferate as procyclic trypomastigotes (Copied from Bartholomeu et al., 2014).

1.2 Current treatment for disease caused by kinetoplastids The current HAT treatment is based on five drugs which were developed decades ago, all of which have been reported to possess adverse side effects, challenges in efficacy, administration, and compliance. As recently reviewed by in Field and colleagues (Field et al., 2017), the first stage of HAT caused by T.b. rhodesiense and T.b. gambiense is treated by and , respectively. However, both of these drugs are administered intraparenterally and are associated with toxicities. is effective against second- stage HAT caused by both T.b. rhodesiense and T.b. gambiense while eflornithine is only active for second-stage HAT caused by T.b. gambiense. The arsenical melarsoprol is administered intravenously for 10 days and is highly toxic causing substantial levels of drug- related mortality due to reactive encephalopathy (Kennedy, 2013). The number of treatment failures after arsenical melarsoprol administration continue to increase due to drug resistance or other unknown factors. The pharmacokinetic studies on melarsoprol led to the treatment regimen being changed to a 10-day course rather than the previous 21- to 35-day course, thus improving patient compliance and reducing hospital costs (Kennedy et al., 2013). Being polyamine biosynthesis inhibitor, eflornithine has been commonly administered for HAT cases caused by with T.b. gambiense, which are resistant to treatment with melarsoprol (Malvy and Chappuis, 2011). The drug is suitable for second stage of disease

4 and is not effective against T.b.rhodesience. It is usually administered over 14 days as four daily intravenous infusions and has less severe adverse reactions compared to melarsoprol. However, administering 56 intravenous infusions present a logistical drawback especially in resource-limited settings. Nifurtimox-eflornithine combination therapy (NECT) have been shown to be effective in cases of second-stage HAT that are either refractory to treatment with melarsoprol alone or in situations where is unavailable. NECT has reduced treatment period and less costly than eflornithine monotherapy (Simarro et al, 2012). Moreover, the lack of paediatric formulations for some of the mentioned drugs together with contraindications for pregnant women and those of childbearing age further limit the use of these drugs. Vaccine development for HAT faces the challenge of parasite’s antigenic variation whereby the parasites make several antigenic variants by alternate expression and recombination of a repertoire of VSG-encoding genes, allowing them to escape the host immune response (LaGreca and Magez, 2011).

Only two drugs, nifurtimox and are currently available for treating Chagas disease. These drugs are orally administered but faces side effects, a long treatment period (>60 days) and variation in sensitives to the parasites (de Castro et al., 2006; Moraes et al., 2014). Both of these drugs are reasonably effective against the acute form of the disease but have poor tolerability and patient compliance (Field et al., 2017). It has been reported elsewhere that once the heart failure develops in chronic Chagas disease, the treatment with benznidazole becomes irrelevant (Wang et al., 2012). Other studies have reported the efficacy of benznidazole as a treatment for adults with Chagas disease who havebeen infected with the parasite for a long time (Viotti et al., 2006 and de Castro et al., 2006). However, the absence of well-structured randomized placebo-controlled studies to assess the specific treatment outcomes in different groups has adversely restricted the use of these drugs. There have been some successes however, with antifungal triazoles and protease inhibitors in experimental models of infection with T. cruzi (Doyle et al., 2007). For visceral leishmaniasis (VL), a liposomal formulation of , AmBisome, was shown to be the most effective and well-tolerated treatment, with a single dose of 5 mg/kg curing 90% of patients (Singh et al., 2012). However, the use of AmBisome as treatment was limited due to the toxicity, requirement for intravenous administration and high costs associated with the drug (Bern et al., 2006; Field et al., 2017). is currently the only oral treatment for VL (Field et al., 2017). However, its clinical use has been limited due to teratogenic effects and increasing reports of treatment failures.

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The pentavalent antimonials, and have also been used to treat both VL and CL. Introducing the generic brands of these drugs helped reduce their purchasing costs. However, these drugs are administered parenteral for up to 30 days, they are cytotoxic, and are now becoming obsolete for treatment due to the emergence of drug-resistant parasites (Singh et al., 2012). The emergences of antimonial-resistant parasites lead to the introduction of the antibiotic amphotericin B, which was previously a second-line drug as a first-line drug for treating leishmaniasis in India. The pentavalent antimonials are considered first-line treatment in combination with in Africa (Field et al., 2017). An aminoglycoside Paromomycin has also been reported as a very effective drug in treating leishmaniasis in India (Sundar et al., 2007). Despite havingefficacy essentially equivalent to that of amphotericin B, Paromomycin is associated with ototoxicity and is intramuscularly administered with pain at site of injection (Field et al., 2017).

1.3 Challenges to research on pathogenic kinetoplastids Although diseases caused by the kinetoplastids continue to disable and kill hundreds of thousands of people in underdeveloped tropical regions, efforts towards identifying effective treatment options continue to receive less attention. The pharmaceutical industry and Western governments have shown little or no interest in supporting research aimed at developing new drugs for kinetoplastid diseases. Perharps this might be because studies on these neglected tropical diseases are associated with little or no prospect of generating significant short- or long-term financial gain. Another possible reason to the slow pace of research on kinetoplastids might be that many fundamental aspects of trypanosome biology have not been studied in depth. Research on pathogenic kinetoplastidshas been mostly hindered by the need to handle these organisms safely in a laboratory, requiring dedicated containment facilities. However, these facilities are very expensive to build, maintain and equip, as experimental apparatus cannot be moved in and out of the containment without rigorous decontamination. In addition, the expensive serum-containing media and difficulties in cultivating these parasites in high yields for different protocols, renders research on kinetoplastids almost impractical and unattractive especially to researchers dwelling in resource limited countries where the kinetoplastid diseases are endemic.

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1.4 C. fasciculata as a model organism to study kinetoplastid biology Protists of the genus Crithidia (: Trypanosomatidae) are flagellate parasites that only infect . The genus Crithidia contains a number of species with a wide host specificity that are able to parasitize a variety of species grouped into the orders Diptera, Hemiptera and Himenoptera. The host specificity of these organisms depends on the species of the parasite. In particularly, C. fasciculata successfully infects many species of mosquitoes (Wallace, 1966). Crithidia parasites exist in many life cycle forms but only two forms are clearly distinguished; the Choanomastigotes and the Amastigotes. Choanoamastigotes are free-swimming stumpy cells that are round in their posterior part and truncated in the apical pole by the funnel- shaped flagellar pocket close to the kinetoplast, which is slightly anterior to the nucleus. Amastigotes are non-motile round cells with a flagellum non-emerging from the cellular body. Although they are extracellular, Crithidia are morphologically similar to amastigotes of the genus Leishmania (Olsen, 1974). The development of C. fasciculata starts in the gut of the culicid, which becomes infected by ingestion of amastigotes voided with faeces of other hosts. In the gut, amastigotes differentiate into choanomastigotes, ensuring proper colonization of the gut. Choanomastigotes later differentiate back into non-motile round amastigotes, which are attached to the gut epithelium by hemidesmosomes frequently leading to damage (Schaub, 1994). Infected adult mosquitoes contaminate aquatic environments with amastigotes as well as flowers when they feed on nectar, thus providing chances for transmission of the parasite. Amastigotes are released within the faeces or the entire body of the dead . Eventually, the larval and pupal instars of mosquitoes get infected in the aquatic habitat and finally amastigotes are transmitted to the adult through the metamorphosing gut leading to completion of the life cycle as shown in Fig 2. Since C. fasciculata has such a monogenetic life cycle involving the extracellular choanomastigote and amastigote stages, they do not infect mammals. This comparison with other species of the same family developing digenetic life cycles responsible for leishmaniasis and trypanosomiasis is of outstanding interest in kinetoplastid research. Unlike trypanosomes and leishmanial species, C. fasciculata is easy to culture in large amounts (>100g wet weight) using inexpensive complex media or fully defined serum-free media (Tetaud et al., 2002). These organisms have been used as feeder cells in the monoxenic culture of Entamoeba histolytica and Malaria parasites (Diamond, 1903).

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Figure 2. The life cycle of C. fasciculata. Amastigotes attached to the culicid gut epithelium are voided in faeces and disseminated in the environment. Eventually, the larval and pupal instars of mosquitoes get infected in the aquatic habitat and finally amastigotes are transmitted to the adult mosquito through the metamorphosing gut. Choanomastigotes are the motile stage that allows the colonization of the gut of the host. GE: gut epithelium. (Copied from Olsen, 1974)

In addition, C. fasciculata parasites are easily amendable to molecular genetics and biochemical analysis and their evolutionary proximity to the pathogenic kinetoplastids makes them a very interesting model organism to study biochemical, cellular, and genetic processes unique to kinetoplastids. The complete genome sequence of C. fasciculate has been determined and is publically available at (http://www.ncbi.nlm.nih.gov/nuccore/440789264) to facilitate genome studies of these organisms. Some examples of genetic, biochemical and cellular processes in which C. fasciculata has been utilised as a model organisms to study trypanosomes and leishmania species are summarised in a Table 2.

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Genetic/Biochemical/cellular process studied Ref. Effect of tunicamycin on the glucose uptake, growth, and Rojas et al., 2014 cellular adhesion Inactivation oftopoisomerase I by Fenton systems Podesta et al., 2003

Identification and characterization of a kinetoplast-specific Sinha et al., 2004 DNA ligase The replication of kinetoplast DNA Liu et al., 2005 Trypanothione synthesis Comini et al., 2005

Trypanosomatid flagellum biogenesis Sahin et al., 2004

Pharmacodynamic screening-medicinal plants Tasanor et al., 2006 Application of magnetically induced hyperthermia as a Grazu et al., 2012 potential therapy against parasitic

kinetoplast DNA organization- The KAP1 protein Lukes et al., 2001 Lipoarabinogalactan structures Schneider et al.,1996 Saxowsky et al., 2002 Mitochondrial DNA repair- Mitochondrial DNA Polymerase β Genetic manipulation Hughes and Simpson, 1986 Expression vector Tetaud et al., 2001

Table 2. Use of C. fasciculata to study various biological processes. Studies highlighting genetic, biochemical and cellular processes in which C. fasciculata has been utilised as a model organism to study trypanosomatids cellular and molecular biology.

1.5 Aims of the study The main aim of this study was to explore the utilization of C. fasciculata as a convenient model organism to study the cell biology and drug discovery vehicle of the pathogenic kinetoplastids.

We specifically aimed to;

i. Develop and validate an expression vector that can be used to express and isolate PTP tagged kinetoplastid proteins in C. fasciculate parasites. ii. Develop a Resazurin-reduction cell viability assay with C. fasciculata and utilise the developed assay to screen for anti-Crithidial compounds from the Open access chemical boxes. iii. Investigate the effects of gamma irradiation on the C. fasciculata parasite’s cell growth, metabolic viability, motility and morphology.

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2. Chapter 2: Developing and validating an expression vector for subsequent isolation of PTP tagged kinetoplastids proteins in C. fasciculata.

2.1 Introduction 2.1.1 Kinetoplastids proteins expression systems In search for new effective drugs against kinetoplastid diseases, potential drug targets need to be identified, characterised and validated. Recent advancements in transformation, protein expression, isolation and purification procedures have been a major technical breakthrough in identification and characterization of novel proteins as drug targets. Highly efficient expression of proteins remain an important alternative to the isolation of protein from native sources and is especially useful when the native protein is normally produced in limited amounts or by sources that are impossible, expensive and/or dangerous to obtain or propagate. In the last few years, gene-transfer systems have been developed specifically for pathogenic and medically important kinetoplastids; Leishmania spp, T.brucei and T. cruzi. The parasites transformation is usually facilitated by integration, which occurs exclusively by homologous recombination, or by episomal shuttle vectors. Moreover, various kinetoplatids proteins expression systems are currently available to facilitate relevant studies. These included; the pTEX vector for rapid expression of proteins in both T. cruzi and Leishmania (Coburn et al., 1991 and Martinez-Calvillo et al., 1997, respectively). Attempts to transform C. fasciculata and T.brucei with pTEX vector however, have been unsuccessful (Coburn et al., 1991). The pX vector, which has been used for stable transformation of Leishmania major and have been successfully applied in studying the parasite’s surface antigen genes (LeBowitz et al., 1990). Of recent is the Lexsy, which has been widely used in expressing proteins in non-human pathogenic Leishmania tarentolae parasites of gecko. The Lexsy system allows not only easy handling like E. coli and yeast, but also full eukaryotic protein folding and the mammalian-type posttranslational modifications of target proteins. The pTSO-HYG4 vectors in T. brucei, which utilize the PARP promoter and is able to replicate extrachromosomally with the aid of minicircle origin of replication have been reported (Sommer et al., 1996). Other T.brucei expression systems include the TetR and the bacteriophage T7RNAP, which can allow transgenes to be highly transcribed and expressed in T.brucei (Wirtz et al., 1999). Mammalian and protozoan signal peptides have also observed to function in T. cruzi to target proteins to different cellular compartments (Garg et

10 al., 1997). In addition, bioactive cytokines (IL-2 and IFN-gamma) have been produced in both T. cruzi and Leishmania (Tobin et al., 1993), suggesting that mammalian signal peptides are recognized and processed by these protozoans. Vectors bearing an rRNA promoter have been constructed by Biebinger and colleague (Biebinger and Clayton, 1996), for expressing foreign genes in C.fasciculata. More recently, the pNUS vectors have been constructed to express biologically active proteins in Crithidia and L. amazonensis (Tetaud et al., 2002). Although a number of expression systems have proven useful for production of various heterologous proteins, none of these systems is universally applicable for the production of all proteins. A protein expression system that provides for the efficient expression and isolation of correctly post- translationally modified heterologous proteins in a non-pathogenic host would therefore constitute a highly desired advance in the art.

2.1.2 Multi protein complexes Most biological processes in a cell are carried out by proteins and by their ability to form multi-protein complexes at an appropriate time. Individual proteins may participate in the formation of a variety of different protein complexes (Gavin et al., 2006). Proteins that are in the same complex can differ in specific function, but they function in the same overall process and hence have a related general function (Panigrahi et al., 2008). Indeed, this protein interaction can regulate proteins activities through either post-translational modification or conformational-transformation. Studies of individual proteins in a multi-protein complex assembly can provide clear information of their specific functions in the complex (Cusick et al., 2005). The interactions of proteins in a complex to control various significant biological processes for cell viability should therefore not be overlooked as a focus of potential drug targets.

2.1.2.1 The replication factor C protein complex Eukaryotic replication factor C (RFC) complex previously known as a clamp loader is a heteropentamer consisting of five essential subunits referred to as RFC1 through RFC5(Bowman et al.,2004). With the exception of the large RFC1 subunit (approximately 128 kDa in humans), the RFC 2, 3, 4 and 5 subunits are approximately 38–41 kDa each (Yao and O'Donnell, 2012). The five subunits contain a region of homology with one another (Erzberger and Berger, 2006). This region of homology defines a large family of proteins

11 referred to as AAA+ proteins. The AAA+ homologous region folds into two domains that bind ATP: the larger N-terminal domain contains the P-loop and DEAD box ATP site motifs, while the smaller C-terminal domain is outside the AAA+ homology region and is mostly composed of α-helix. The C terminal domain mediates a strong inter-subunit interaction that holds the pentamer tight (Bowman et al., 2004). The five RFC components are organised in a circular form (Fig.3a). The C-terminal domains define a nearly planar circle referred to as a “collar domain” as shown in Fig 3a. One component of RFC is shown (Fig. 3c), to illustrate the three domains structure of clamp loader subunits. By convention, clamp loaders are viewed from the “side” as C-terminal domain on the top, and the N terminal AAA+ domains on the bottom. Proceeding counterclockwise around the circle from the subunit at the far right, the subunit positions are read as A- E subunits or simply RFC1-RFC5 (Fig. 3b and c). The collar C-terminal domain forms a tightly closed circle with no gap and holds the complex together. In all eukaryotesclamp loaders, a gap exists in between the AAA+ domains of subunits RFC1 and RFC5 as shown in Fig. 3a and c. This is so because RFC1 has a C- terminal region that extends across the gap and protrudes down toward the N-terminal face of the clamp loader and it interacts directly with the Proliferating Cell Nuclear Antigen (PCNA) and with DNA (Fig. 3c) (Bowman et al., 2004).

Figure 3. Architecture of the RFC complex: The five subunits of the RFC complex are referred to as RFC-A, RFC-B, RFC-C, RFC-D and RFC-E, respectively, moving in a right- handed sense around the assembly, with the thumb pointing towards the collar (a and b). The three domains of RFC and the extensions of C-terminal domain that interact with PCNA (c). The yeast and human nomenclature for each subunit is shown in parentheses (copied from Bowman et al., 2004).

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The RFC complex functions are universally conserved in all eukaryotic organisms. As clamp loaders, RFC complexes catalyses the process of loading the PCNA on to the 3` ends of nascent DNA strands. As described by Hedglin and his colleagues (Hedglin et al., 2013), the complex utilizes the ATP and act as a protein topoisomerase to open the ring of PCNA so that it can encircle the DNA. The ATP hydrolysis causes release of RFC, with concomitant clamp loading onto DNA thus permitting highly processive DNA replication (Fig. 4)

Figure 4. Schematic model of eukaryotic RFC complex as clamp loader. ATP binding to RFC enables RFC to bind and open PCNA. RFC places PCNA onto DNA and then hydrolyses ATP to eject itself out of the PCNA-DNA complex (copied from Hedglinet al., 2013).

The RFC complex have also been reported for switching DNA polymerase enzymes from DNA Pol α to Pol δ during initiation of leading strand DNA replication in the Simian virus 40 origins of replication and as well as synthesizing of Okazaki fragments during lagging strand DNA replication (Yao and O’Donnell, 2012). As a structure specific DNA binding protein, RFC complex is a primer recognition factor for DNA polymerases especially δ and ɛ, and function as accessory proteins for these enzymes (Bowman et al., 2004). The complex binds primers synthesized by Pol α-primase blocking them for further elongation and increasing their affinity to Pol δ.

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Other studies have reported the requirement of RFC complex in DNA repair mechanisms and in replication checkpoints in yeast cells (Schmidt et al., 2001; Chen et al., 2009). When there is no DNA replication due to DNA damage, checkpoint regulatory networks are induced by RFC complex, which avoid the cells to enter in mitosis (so cells accumulate in the interphase) until the DNA damage is restored(Chen et al., 2009).

Alternative forms of RFC protein complexes or RFC-like complexes (RLC) have been purified in various organisms such as yeast, plants and mammals. Each RLC is made up of the four small subunits of the RFC (RFC2-5), but with a specific large subunit of that complex (Fig. 5). One such RLC is Elg1-RFC complex in yeast and humans in which Elg1 replaces RFC1 (Fig.5a) (Kim and MacNeill, 2003). Cells with Elg1-RLC deletions had chromosomal instabilities and showed slow S-phase progression suggesting the role of Elg1- RFC in genome stability. A second form of RFC is Rad24–RLC in which the RFC1 subunit is replaced by Rad24 (known as Rad17 in humans) resulting in the formation of the pentameric Rad24–RLC (Fig.5b), that specifically loads the heterotrimeric Rad9–Rad1–Hus1 (9-1-1) clamp complex onto DNA (Majka and Burgers, 2003). In addition, the 9-1-1 complex comprises of other proteins such as Ddc1 and Mec 3 (Kim and MacNeill, 2003). The 9-1-1 clamp complex activates the DNA damage checkpoints (G1, G2 and in the intra-S phases) to ensure that cell cycle progression is halted until repair is complete (Eichinger and Jentsch, 2011). The third form of alternative RFC in is the Ctf18-Dcc1-Ctf8/ Ctf18-RLC complex (Fig.5c). Similar to the other two forms, the RFC1 subunit is replaced by Ctf18 to form the heteroheptameric Ctf18–RLC complex. The Ctf18 subunit is known to interact strongly with two other subunits; the Dcc1, and Ctf8. The Ctf18, Dcc1, and Ctf8 together form a complex (Ctf18-Dcc1-Ctf8) with the four small subunits of RFC (Mayer et al., 2001). Unlike other RLC, the Ctf18-RLC complex is known to play crucial role in chromosome cohesion, the process by which newly replicated sister chromatids remain physically associated until mitotic anaphase (Lengronne et al., 2006). Apart from helping the replicated chromosomes stay together until mitosis, the Ctf18-RFC complex also enhances genome stability in yeast (Ansbach et al., 2008; Gellon et al., 2011). This is achieved by its ability to help the DNA replication machinery move through triplet repeats and able to repair any resulting DNA damage.

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Figure 5. Schematic representation of the three RLCs in eukaryotes with their cognate sliding clamps. The complexes are defined by their large subunits, as the four small RFC subunits designated 2–5 are common to all. Note that the large subunits possess extended amino- and carboxy-terminal regions. (a) Elg1-RLC where RFC1 is replaced by Elg1,(b) Rad24-RLC in which RFC1 is replaced by Rad 24 (Rad17) and (c) Ctf18-RLC in which RFC1 is replaced by Ctf18 but has two additional non-RFC subunits Ctf8 and Dcc1 (Copied fromKim and MacNeill, 2003).

2.1.2.2 The RNA exosome protein complex As described by Makino and Conti, the core of eukaryotic exosome complex is made up of six RNase PHlike subunits, which assemble into a ring-like structure and three additional subunits composed of S1/KH domains (so-called cap proteins), forming a coaxial ring (Makino and Conti, 2013). At the base of the complex are the six RNase PH-like proteins (Fig. 6): RRP41, RRP42, RRP43, RRP45, RRP46, and Mtr3, forming a core ring structure and central channel of the complex. On top of the complex are the three catalytic “cap” subunits; RRP4, RRP40, and Csl4, which have the S1-RNA binding domain (SI) and the K- homology (KH) domain. Apart from the nine core exosome components, two extra subunits- -RRP6 and RRP44/Dis3 have been reported in yeast (Lorentzen et al., 2008a). The RRP44/Dis3 is a hydrolytic processive exoribonuclease subunit and is not available in the archaeal organisms (Navarro et al., 2008). If RRP44/Dis3 is associated with humans or trypanosome exosomes, the interaction is weak because it has never been purified in these organisms (Chen et al., 2001; Estevez et al., 2001; Estevez et al., 2003).

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Figure 6. (a) A simplified architecture of Saccharomyces cerevisiae exosome complex and (b) its model interaction with the mRNA fragment (Copied from Navarro et al., 2008).

The exosome complex plays a very significant role in RNA metabolism processes. In the nucleus, the exosome complex regulates messenger RNA numbers during their turnover processes and participates in various RNA maturation processes (Makino and Conti, 2013). The complex degrades the RNA maturation by-products such as 5’ External Transcribed Sequences and functions in RNA quality control processes by destroying aberrant products of rRNA, tRNA,snRNA and snoRNA in the nucleus (Fig. 7b) (Tomeckiet al., 2010). In cytoplasm, the complex participates in RNA surveillance systems (“non-stop decay”, which degrades mRNA with a no stop codon, “nonsense-mediated decay”, which degrades products possessing immature stop codons and the “no-go decay”, which degrades RNA within stalled ribosomes). The exosome complex has also reported to degrade the 5′ intermediate segments from the RNA interference processes in the both nucleus and cytoplasm (Tomeckiet al., 2010).

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Figure 7. Schematic examples of eukaryotic exosome complex catalytic activities. (a) Single stranded substrates with enough long single stranded overhangs are recognised by the trimetric cap of RNA-binding proteins and passes through the central channel of the complex before accessing the RRP44/Dis3-exoribonuclease active site. (b) RNA substrates with secondary structures but of no 3’ single stranded extensions of enough length to pass through the central channel, access the RRP44/Dis3 exoribonuclease active site directly after being recognized by the OB-fold RNA-binding domains. The PIN domain can endonucleolytically cleavage (yellow scissor) single stranded loops of the secondary structure to assist in 3’-5’ exoribonucleolytic degradation by RNB domain catalytic activity. (c) In the nucleus, transcripts polyadenylated by the TRAMP are trimmed by RRP6 without passing the central channel and later get degraded (Copied fromTomeckiet al., 2010)

2.1.3 Overview of protein complexes purification methods Although several biochemical studies have identified proteins likely to play a role in the establishment and maintenance of the pathogenic kinetoplastids’s life cycle, specific functions of these proteins has remain unknown in several cases. The process of isolating and identifying protein complexes is very useful when the purpose is to gain more understanding on the specific functions of individual proteins in the complex as well as to determine their importance at a molecular level. Complete genome sequencing of various microorganisms has created a chance to analyse roles of various proteins encoded in their genes. More knowledge on various cellular processes can be acquired through analysing, characterizing and identifying interactions, which exist among proteins in the complex (Schwikowski et al., 2001). The two-hybrid systems were commonly used identity components of specific protein complexes. However, these were time consuming, labour intensive and limited to a small- scale protein analysis. Moreover, the two-hybrid systems have low rates of true positives and

17 true negatives results. More effective and reliable strategies of studying protein-protein interactions have been highly needed. With the development of the Tandem affinity purification (TAP) coupled to Mass spectrometry over the past decades, many proteins complexes have been isolated and the interacting partners identified. As long as a specific protein complex of interest is effectively and adequately isolated, proteins within the complex can be recognised and characterised by TAP-mass spectrometry. Enhanced by the available databases of complete genomic sequences of various organisms, though initially designed for yeast cells, this approach has been widely employed in various organisms (Puig et al., 2001) to facilitate the identification and characterisation of their respective proteins. As suggested by Schimanski et al (2005), TAP-mass spectrometry limitations are observed in the protein isolation process rather than in the process of identifying them. This is mostly because an individual protein may possess different characteristics from other associated proteins in the complex. More recently, the tagging of protein of interest with either peptides or protein domains has seemed to be a promising strategy of bypassing this limitation during purification process. The conventional TAP methods required the fusing of a C-terminal TAP tag to a protein of interest. The TAP tag composed of two IgG binding units of protein A of S.aureus (ProtA) and a Calmodulin binding domain (CBP) with a cleavage site for a Tobacco etch virus (TEV) protease inserted between them (Rigaut et al., 1999). Apart from a C-terminal TAP tag, there is an N-terminal TAP tag, which is a reverse orientation of the C-terminal TAP tag (Fig. 8a). Two TAP steps are required to purify the cell extracts of the tagged protein of interest after transformation as shown (Fig.8b).

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Figure8. A schematic representation of TAP tagging and an outline of the purification procedure. (a)Representations of a C- and N-terminal TAP showing orientation of Protein A domains and CBP. (b) The TAP purification procedure. In the first step, the protein complex containing the tagged protein of interest binds to IgG matrix by the ProtA fraction. The protein complex is then eluted using TEV protease under native conditions. In the second step, the elution fraction of the first purification step is incubated with beads coated by calmodulin in the presence of calcium. Subsequently, contaminants and the remainder of TEV protease used in the first step are eliminated through washing. Finally, the protein of interest in a complex is obtained by elution using EGTA. (Copied from Rigaut et al., 1999).

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Compared to the traditional protein purification methods, the TAP method has been observed to be a very effective tool for isolating pure proteins in adequate quantities from a small volume of cell culture and under native conditions to retain their functions (Rigaut et al. (1999). However, although this has been the case, the TAP tool has been unsuccessful in various cicumstances. For instance, Gavin and his collegues reported the failure of the method to identify and isolate the tagged and associated proteins in yeast (Gavin et al., (2001). The failure was claimed to be due to the TAP tag which interfered with the function of the protein, its location and as well as complex formation. Recently, several new TAP tags combinations have been developed to overcome the limitations of the conventional TAP tags, including PTP (for ProtC-TEV-ProtA) where CBP is replaced by protein C epitope (ProtC) (Fig. 9b), reducing the overall size of the tag from 184 to 169 amino acids and the molecular mass from 20.6 to 18.9 kDa. After TEV protease cleavage, 44 and 29 amino acids accounting for 5.1 and 3.4 kDa, respectively, remain on TAP- and PTP tagged proteins. ProtC is derived from human protein C, a vitamin K- dependent plasma zymogen specifically expressed in hepatocytes. The monoclonal antibody HPC4 recognizes this epitope with high affinity and, as a unique property, has a calcium- binding site that needs to be occupied for its interaction with ProtC.

Figure 9. A schematic representation of TAP and PTP tag. (a) TAP tag showing Protein A domains, TEV and CBP. (b) PTP tag where CBP is replaced by Prot C (Copied from Schimanski et al., 2005).

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2.2 Materials and methods 2.2.1 Organisms and reagents The C. fasciculata promastigotes clone HS6 were grown at 27°C with gentle agitation in serum-free defined media as described in Appendix 1). The media pH was adjusted to 8.0 with either NaOH or HCl before haemin (10µg/ml) was added. The parasites' genomic DNA was extracted according to DNA extraction procedure described by Bernards et al (1981) as described in Appendix 2. Conventional DNA manipulation procedures were conducted using DH5α competent E.coli cells. All chemicals and reagents used were of the highest grade and were purchased from Sigma-Aldrich or Thermo Fisher Scientific. DNA manipulation enzymes were purchased from either New England Biolabs, Thermo Fisher Scientific or Promega.

2.2.2 General Molecular biology techniques 2.2.2.1 Designing primers and amplifying ORFs of target proteins Reverse and forward primers were designed to amplify the entire ORFs of our target subunits (RFC3 and RRP4) from the C. fasciculata genomic DNA. Briefly, 10 random bases (GGTGGTGGTG) and 21 consecutive bases of the target gene sequence after a start codon were added upstream and downstream NdeI sequence (CATATG) respectively, to create a 5’ NdeI (forward) primers. The 3’NotI (Reverse) primers were also designed by adding 10 random bases (GGTGGTGGTG) and 24 consecutive bases of the target gene sequence before a stop codon (in reverse complement) upstream and downstream the NotI sequence (GCGGCCGCC), respectively. Oligonucleotides were then ordered from Integrated DNA

Technologies. All primers had melting temperatures (Tm) between 65°C and 74°C, the GC content between 40 and 60%, with balanced distribution of GC-rich and AT-rich domains without secondary structures and had no recognisable intra-primer homology or inter-primer homology. The designed primers are shown in Appendix 3. A conventional PCR was used to amplify the ORFs of the target protein subunits (RFC3 and RRP4) using Q5 High-Fidelity DNA polymerase following the manufacturer’s instructions with slight modifications as described in Appendix 4a.

2.2.2.2 Agarose-gel electrophoresis The purified DNA products were mixed with 10x loading dye and loaded onto a 1% agarose gel (1% agarose in 1x TAE and 0.001% ethidium bromide) alongside 0.5 µg of a 1 kb DNA

21 ladder (Fermentas). Gels were run at 120 V for 45 minutes and the DNA was visualised using a UV transilluminator (Syngene U Genius).

2.2.2.3 Preparative restriction digest and Ligations for cloning Prior to cloning, the vector pNUS-PTPcH and inserts (RFC3 and RRP4) were digested in 60µl reactions with restriction enzymes NotI and NdeI (NEB) for 3 hours at 37°C and dephosphorylated during the last 60 minutes with Antarctic phosphatase (NEB) according to the manufacture’s instruction. The DNA fragments were gel purified using Thermo Scientific DNA purification kit following the manufacture’s instruction. 10% of the eluted volume was resolved in a 1% agarose gel containing 0.001% ethidium bromide to confirm the presence and size of the fragments before ligations. For each ligation reaction, 50ng of the gel-purified vectors were combined with 3-fold molar excess of the insert (calculated using NEBioCalculator) and the final volume adjusted to 10µl with distilled sterile water. 10µl of 2x Quick ligation buffer followed by a 1µl of the Quick T4 DNA ligase was then added and mixed thoroughly and briefly centrifuged before incubation at room temperature (25°C) for 20 minutes. The ligation products were directly used for E.coli transformation. Protocols and recipes for preparative restriction digests, diagnostic digests, ligations and agarose electrophoresis are described in Appendix 5.

2.2.2.4 E.coli transformation and Isolation of plasmid DNA Conventional TSB transformation procedures were used as previously described in Inoue et al. (1990). Briefly, 20ml of LB medium was inoculated with 100µl of the over night grown E.coli DH5α cells and incubated at 37°C for 4 hours in a shaker incubator. Cell culture (optical density ~0.5) was then spun down at 3,000 rpm for 10 minutes to pellet cells. The pelleted cells were suspended in 1 ml of TSB (See recipe in Appendix 6) and thawed on ice for 30 minutes to make them competent. For each transformation reaction, 100 µl of the competent cells were transferred to a chilled microcentrifuge tube on ice. To this, 10µl of the plasmid DNA was added and the mixture kept on ice for 30 minutes. After 30 minutes incubation on ice, 200µl of LB media was added and the mixture was later incubated at 37°C with shaking for 1 hour. Later, 150 µl of cells were plated on LB plates containing 100µg/ml ampicillin using glass beads and incubated over night at 37°C.

2.2.2.5 Colony PCR for transformations and restriction digest Transformed cells were analysed by a standard PCR technique by determining the presence of the insert DNA in the plasmid construct using MyTaqRedDNA polymerase following a

22 protocol described in Appendix 4b. Briefly, a final reaction mixture (20µl) contained 5µl volume of cells (from 100µl colony suspension) to provide DNA template during initial PCR heating step, 5µl of the insert specific primers (4µM each) and 10µl MyTaqRed Mix. The presence of a PCR amplicon and size of the product were determined by 1% agarose gel electrophoresis. A miniprep culture was prepared by inoculating a 50µl of correct transformed cells colony suspension in 5ml of LB media containing ampicillin (100µg/ml) for isolation and incubated overnight in a shaker incubator. The Plasmid DNA was purified using a Thermo scientific Miniprep kit following manufactures instructions. A portion of the eluted volume containing plasmids DNA were digested in 20µl reactions with NdeI and NotI enzymes according to manufactures instruction and as described in Appendix 5, to screen and confirm if the plasmids contained the correct inserts. The purified plasmids at a concentration of 1.2µg DNA in 60µl dH2O and the associated primers (50µl of 3.2 µM) were then sent for sequencing at Dundee sequencing services centre to verify the fidelity of cloning process.

2.2.3 Construction of the expression vector (pNUS-PTPcH) The expression vector pNUS-PTPcH was constructed by S. MacNeill (personal communication). Briefly, part of pNUS-SPnHc EcoRI fragment (102 bp) encoding a signal peptide (Tetaud et al., 2001), was removed and replaced with sequences derived by PCR amplification of the PTP tag-encoding region of plasmid pC-PTP from Schimanski and his colleagues (Schimanski et al., 2005). The cloned region lacked the AflII, EcoRI and BamHI sites internal to the PTP tag sequence that are found in pC-PTP and includes unique restriction sites for NdeI, XhoI, EcoRV and NotI upstream of PTP for fusing target sequences. The full sequence of the pNUS-PTPcH vector is shown in Appendix 6.

2.2.4 Identification of C. fasciculata and T. brucei homologous proteins from yeast RFC and exosome complexes Protein sequences of specific known subunits of yeast Saccharomyces cerevisiae’s replication factor C and the exosome complexes were used to BLAST search the TriTrypDB database to identify putative C. fasciculata and T. brucei homologues. The online BLASTP program was used to determine the percentage identity between the protein sequences.

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2.2.5 Cloning of the target subunits Oligonucleotides (Appendix 2) were designed to amplify the entire ORFs of Cf-RFC3 and Cf-RRP4 from the C. fasciculata genomic DNA. Using conventional PCR (Appendix 4a), fragments of Cf-RFC3 and Cf-RRP4 were cloned in-frame with the PTP tag coding sequence of the constructed pNUSPTPcH vector using NdeI and NotI restriction enzymes, to create C- terminal PTP-tagged versions pNUS-RFC3-PTPcH and pNUS-RRP4-PTPcH. The TSB transformation method was used to propagate the plasmids into DH5α E.coli competent cells and the plasmids DNA was isolated as previously described. To verify the fidelity of the cloning process, the purified plasmid DNA were sequenced using pC-Seq-F and pC-Seq-R (Appendix 3) as described in Sanger et al. (1977).

2.2.6 Parasite transfection and generation of cell lines For a successful transfection, C. fasciculata parasites were grown to a log phase at 27°C in a shaker incubator and harvested at a density of ~1 x 107 cells/ml by centrifugation at 3000 rpm for 5 minutes. The cell pellet (~2 x 107 cells/ml) was suspended in 100µl of human T-cell Nucleofactor solution (Lonza) and transferred to a 0.4 cm cuvette containing 15-60µg of purified pNUS-RFC3-PTP or pNUS-RRP4-PTP supercoiled plasmids DNA. The mixture was then subjected to program X-014 of the Amaxa electroporation system (Burcard et al., 2007). Electroporation cells were left on ice for 5 minutes and transferred into a culture flask containing 5ml of fresh medium and incubated at 27°C to recover. After 24 hours of recovery, the cell culture was supplemented with 5 ml of fresh medium followed by hygromycin final concentration of 25µg/ml. Hygromycin-resistant cell lines were subsequently grown with at least 50 µg/mlof drug and viable clones observed within 5 to 10 days. Resistant cell lines were maintained by supplementing the culture with fresh medium containing the antibiotic.

2.2.7 Expression and PTP purification of the proteins To confirm if the selected cell lines expressed the PTP fusion proteins, cell lysates were prepared from the parasites and analysed by SDS-PAGE and Western blots. Briefly, a sample volume of 1ml (~1 x 107 cells) was harvested by centrifugation at 2000 g for 5 minutes. The cell pellet was heated to boil in 2 X SDS-PAGE sample buffer at 95oC for 5 minutes. The protein extract sample was resolved in a 12 % SDS-PAGE and blotted on a Polyvinylidene difluoride (PVDF) membrane. To detect PTP fusion proteins, blots were incubated with PAP reagent, which contained primary antibodies raised against Protein A epitopes of the PTP tag, diluted 1:2000 using 5% milk-PBS-Tween (0.05%) blocking solution. Blots were developed using Goat-Anti-rabbit IgG (H+L DyLightTM 680) conjugated secondary antibody

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(ThermoFisher Scientific) diluted in 1:10000 according to the manufacturer’s instructions and scaned on Odyssey scanner using the 700nm channel.

Purification of the target proteins and their interacting partners in the complexes was conducted according to the generic TAP method described inSchimanski et al. (2005) with slight modifications. Recipes for solutions and buffers used in the purification experiments are described in Appendix 7. Briefly, a volume of 2.5 litres of culture (~ 2x107 cells/ml) was harvested by centrifugation at 800 g for 10 minutes. The cell pellet was washed three times with 5 ml PBS to a final packed cell volume of approximately 4 ml. The cell extract had a volume of ~6.5 ml and contained 150 mM sucrose, 300 mM potassium chloride, 40 mM potassium L-glutamate, 3 mMMgCl2, 20 mM HEPES-KOH (pH 7.7), 2 mM dithiothreitol, 0.1% Tween 20, and half of a Complete Mini EDTA-free protease inhibitor cocktail tablet (Roche, Indianapolis, IN). Cells were Dounced in continuous strokes for 5 minutes in a cold room using a 7 ml Dounce homogenizer (Sigma-Aldrich) and centrifuged at 20,500 g for 10 minutes at 4°C. For IgG affinity chromatography, the resultant lysate was filtered straight into a 10 ml Poly-prep chromatography column (Bio-Rad, Hercules, CA) containing a volume of 200 µl of PA-150 buffer equilibrated IgG Sepharose beads (GE Healthcare). The top and the bottom of the column were sealed with Parafilm and the column rotated for 2 hours at 4°C allowing the PTP tagged protein to bind to the IgG beads. The beads were later washed 2 x with 10 ml PA-150 before equilibrating the column with 8ml TEV buffer. To TEV cleave the IgG matrix bound proteins, a 20µl of TEV protease was diluted in to 2 mL TEV buffer and added to the column rotating it overnight at 4°C. The TEV and column dead-volume were eluted by washing the IgG beads with 4 ml of PC- 150 buffer. 0.5ml of the remaining Mini EDTA-free protease inhibitor tablet (Roche) and 7.5

µl of 1 M CaCl2 were added to the eluate mixture to avoid any proteolysis in the eluate. The mixture was added to a second equilibrated Poly-prep column containing a volume of 200 µl anti-ProtC affinity matrix beads (Roche) and was rotated for 2 hrs in the cold room to allow the tagged protein to bind to the anti-ProtC affinity matrix. After washing the anti-ProtC affinity matrix beads for 6 times with 10 ml PC-150 buffer, the PTP tagged proteins were eluted with a 1.8 ml EGTA/EDTA buffer at room temperature.

To concentrate the eluted proteins, eluates were bound to a volume of 30 µl of StrataClean resin beads (Stratagene) and pelleted at 5,000 g for 1 minute. The beads were later re- suspended in 20 µl 4XNuPAGE LDS sample buffer and boiled at 95°C to release the proteins. A 20 µl of the sample was loaded onto a NuPAGE 4-12% Bis-Tris pre-cast gel and the

25 proteins were resolved by SDS-PAGE before stained with SYPRO Ruby stain. The stained gels were visualised using a UV transilluminator.

2.2.8 Mass Spectroscopy analysis Individual protein bands were excised from the gels and analysed by our in-house Mass Spectroscopy and proteomics facility at the University of St Andrews, Biomedical Sciences Research Complex. Briefly, proteins were digested overnight with trypsin prior to separation by AB Sciex 4800 MALDI (Matrix-Assisted Laser Desorption/Ionisation) TOF/TOFTM analyser (AB Sciex, UK). The obtained data were processed with MASCOT and compared against the C. fasciculata or NCBI protein databases to unambiguously identify the proteins.

2.3 Results and Discussion 2.3.1 Identification of C. fasciculata and T.brucei homologous proteins from yeast RFC and exosome complexes

Protein sequences of specific known subunits of yeast S. cerevisiae replication factor C and the exosome complexes were used to BLAST search the TriTryp database to identify putative C. fasciculata and T. brucei homologues. The S. cerevisiae RFC and exosome complexes homologs of C. fasciculata and T. brucei identified are shown in Table 3. The percentage (%) identity between the protein sequences were determined using the NCBI online BLASTP program. The identified homologs were selected based on their maximum identity and E- scores in TriTryp database.

The C. fasciculata RFC and exosome subunits shared around 30-55% and 28-56% sequence identity with the yeast counterparts, respectively and also shared 65-73% and 34-60% sequence identity with the T.brucei homologs, respectively. As expected, C. fasciculata proteins were more homologous to the T.brucei than the yeast counterparts. The RFC complex subunits of the C. fasciculata and T.brucei were more identical compared to the exosomes subunits (average identity of 70% and 45% for RFC subunits and exosome, respectively, between C. fasciculata and T. brucei homologues). This is not surprising as ribosomal RNA metabolisms in trypanosomes are unique as observed in various studies (Ullu et al., 1996; Hartshorne and Toyofuku 1999; Di Noia et al., 2000).

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S. cerevisiae T. brucei homologs C. fasciculata homologs subunits Name Name Gene ID Gene ID Mwt (Da) % Identity %Identity(pos (positives %) itives %) to to S.cerevisiae T.brucei Exosome subunits Exosome RFC RFC1 TbRFC1 Tb927.11. CFAC1_210019 72,644 30 (47) 65 (79)

5650 200 complex complex RFC2 TbRFC2 Tb927.6.3 CFAC1_260050 38,286 44 (66) 69 (83) 890 500 RFC3 TbRFC3 Tb927.9.1 CFAC1_300082 39,856 43 (62) 72 (83)

subunits

2300 900 RFC4 TbRFC4 Tb927.11. CFAC1_230046 33,546 55 (74) 69 (81) 9550 600

RFC5 TbRFC5 Tb927.10. CFAC1_280077 39,086 39 (61) 73 (86) 7990 100

RRP4p TbRRP4 Tb927.7.4 CFAC1_110005 32,088 37 (56) 51 (67) 670 300 RRP6p TbRRP6 Tb927.4.1 CFAC1_290060 80,085 31(48) 51 (65) 630 300 RRP40p TbRRP40 Tb927.9.7 CFAC1_030007 34,302 28 (38) 46 (59) 070 200 Exosome RRP41p TbRRP41 Tb927.10. CFAC1_280032 26,798 48(76) 60 (75) A 7450 800 RRP42p EAP1 Tb927.1.2 CFAC1_170027 43,178 29(61) 41 (53)

complex 580 500 RRP43p EAP2 Tb927.11. CFAC1_300052 32,147 26(43) 40 (56) 16600 700

RRP45p TbRRP45 Tb927.6.6 CFAC1_240024 39,758 31 (49) 46 (62) subunits 70 400 RRP46p TbRRP41 Tb927.2.2 CFAC1_160013 35,618 22(40) 37(50) B 180 900 RRP47 EAP3 Tb927.7.5 CFAC1_180026 23,121 27(50) 34 (48) 460 700 Mtr3 EAP4 Tb927.11. CFAC1_280054 23,286 32 (46) 50 (66) 11030 900

CSL4p TbCSL4 Tb927.5.1 CFAC1_150031 27,882 30 (44) 41 (53) 200 200

Table 3. C. fasciculata and T.brucei homologs of S. cerevisiae’s RFC and Exosome complex subunits. The yeast S. cerevisiae protein sequences were used to BLAST search the TriTrypDB database to identify putative C. fasciculata and T. brucei homologs. The systematic identities, the molecular masses (Mwt) of the predicted C. fasciculata peptides and the percentage of identity are indicated.

2.3.2 Construction of the expression vector TAP has been used for many years as a rapid and efficient method of isolating epitope-tagged protein complexes from crude extracts under native conditions. Initially established in yeasts, the method is now applied to other organisms such as trypanosomes. However, a number of studies have reported the inefficiencies of the original TAP method, which is based on fusing the proteins of interests to a TAP tag consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP) (Schimanski et al., 2005; Drakes et al., 2005; Palfi et al., 2005). In most cases, the protein yield recovery has

27 been very low, an obstacle which has been mainly attributed to the calmodulin affinity purification step. To overcome this limitation, CBP has recently been replaced with PTP and successful results have been reported (Schimanski et al., 2003; Drakes et al., 2005; Palfi et al., 2005; Schimanski et al., 2005). Recently, series of shuttle vectors have been developed by Tetaud and colleagues to facilitate the expression of histidine tagged proteins in C. fasciculata (Tetaud et al., 2002). Extending this work, we modified the initial vector and developed a pNUS-PTPcH vector, which can be utilised to facilitate the expression and isolation of PTP tagged kinetoplastids proteins in these convenient parasites. Briefly, the vector pNUS-PTPcH (Fig. 10c) was constructed by S. MacNeill (personal communication), by removing and replacing the EcoRI fragment (102 bp) encoding a signal peptide in pNUS- SPnHc plasmid (Fig.10a) (from Tetaud et al., 2002) with sequences derived by PCR amplification of the PTP tag-encoding region of plasmid pC-PTP (Fig. 10b) (Schimanski et al., 2005). The cloned region included unique restriction sites for NdeI, XhoI, EcoRV andNotI upstream of PTP for fusing target sequences to the tag (Fig. 10c). As in the original vectors, bonafide replication and transcription promoters were maintained to facilitate episomal expression of the construct. The 5`- and 3`-untranslated (UTR) sequences in the intergenic region of the C. fasciculata phosphoglycerate kinase genes A and B (IG-PGKAB) and the C. fasciculata glutathionylspermidine synthetase(GSPS) 3` UTR were also maintained as in Tetaud et al. (2002), to allow expression and maturation of the target genes and the hygromycin resistant gene.

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Figure 10. Circular maps of the plasmid constructs. (a) The pNUS-SPnH (copied from Tetaud et al.,2002) showing the region encoding signal peptide which was removed and replaced by a PTP tag-encoding sequence from pC-PTP-NEO plasmid (b) (Copied from Schimanski et al., 2005). (c)The constructed pNUS-PTPcH plasmid showing the IG-PGKAB, GSPS and specific restriction sites NdeI and NotI for cloning genes of interest.

2.3.3 Cloning of C. fasciculata RFC3 and RRP4 subunits in pNUS-PTPcH plasmid and Transfection.

Using PCR, we successfully cloned the ORF of target subunits RFC3 (CFAC1_300082900) and RRP4 (CFAC1_110005300) in frame with the PTP sequence of the constructed pNUS- PTPcH shuttle vector to create C-terminal PTP-tagged versions pNUS-RFC3-PTPcH (Fig. 11a) and pNUS-RRP4-PTPcH (Fig. 11b). Unlike the RFC complex, exosome complex have previously been TAP isolated using RRP4 subunit as a bait (Estevez et al., 2001). We therefore took advantage of this approach and also PTP tagged RRP4 in our constructs for validation.

C. fasciculata parasites were succesfuly transfected with the constructed plasmids using Amaxa program X-014 (Burcard et al., 2007), where it conferred hygromycin resistance and persisted as circular extrachromosomal DNAs that could be recovered back in E.coli.

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Figure 11. Circular maps of RFC3 and RRP4 tagged versions. (a) pNUS-RFC3-PTPcH and (b) pNUS-RRP4-PTPcH.In each case, fragments of the target genes, the PTP cassette, the C. fasciculata IG-PGKAB gene flank, the resistance marker and the 3`flank GSPS are drawn in different colours.

2.3.4 Expression and PTP purification of the proteins To validate the pNUS-PTPcH plasmid, we first determined if the cloned genes were expressed in C. fasciculata cells. Cell lysates were prepared from the resistant cell lines and analysed by SDS-PAGE and Western blots. The immunoblot revealed two proteins of sizes ~51kDa and ~57kDa that were confirmed with the theoretical masses as PTP-tagged RRP4 and RFC3, respectively (Fig. 12a). When the hygromycin concentration was increased from 50µg/ml to 200µg/ml, the expression of the tagged proteins also increased though cells grew more slowly (data not shown), a similar observation to (Tetaud et al., 2002) and others. However, increasing the hygromycin concentration to 200µg/ml did not significantly increase the expression of PTP tag protein from empty pNUS-PTPcH cell lines.

To determine if the tagged proteins and their interacting partners could be isolated from their respective complexes, the two consecutive step TAP method was used as previously described in the methods and the eluted complexes analysed by SDS-PAGE and SYPRO- ruby staining. The specificity of the purification method was monitored by generating a cell line expressing the empty pNUS-PTPcH. In this case, no bands were detected from the cell lysates of cells expressing the empty plasmid (data not shown).

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Figure 12. Western blot analysis of C. fasciculata expressing the tagged proteins and Sypro Ruby stained SDS-PAGE gels of TAP purified proteins. (a) 1 x 107 cells/ml were analysed with the PAP reagent directed against the Prot A domains of PTP. Lane 1, C. fasciculata WT; Lane 2, C. fasciculata expressing pNUS-RRP4-PTP and Lane 3, C. fasciculata expressing pNUS-RFC3-PTPcH. Purified components of C. fasciculata Replication factor C (a) and exosome (b) multiprotein complexes, separated by SDS-PAGE and Sypro Ruby stained. The probable identities of subunits as determined by mass spectrometry are indicated. The C. fasciculata cell line expressing the empty pNUS-PTPcH was used as our purification specificity control (TAP-control).

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Table 4a. Mass spectrometric identification of subunits in pNUS-RFC3-PTPcH pull-down Protein Gene ID Mascot score Coverage

RFC1 CFAC1_210019200 586 21% RFC2 CFAC1_260050500 1144 59% RFC3 CFAC1_300082900 1507 77% RFC4 CFAC1_230046600 941 70% RFC5 CFAC1_280077100 997 52% Rad17 CFAC1_260035800 174 6%

Table 4b. Mass spectrometric identification of subunits in pNUS-RRP4-PTPcH pull- down Protein Gene ID Mascot score Coverage RRP41A CFAC1_280032800 988 83% RRP41B CFAC1_160013900 326 34% RRP6 CFAC1_290060300 1943 62% EAP1 CFAC1_170027500 950 34% RRP45 CFAC1_240024400 829 31% RRP40 CFAC1_030007200 1173 70% RRP4 CFAC1_110005300 1330 48% CSL4 CFAC1_150031200 573 40% EAP2 CFAC1_300052700 585 33% EAP4 CFAC1_280054900 691 40%

Five major polypeptide bands were identified in the pNUS-RFC3-PTP pull downs. Mass spectrometry identification of these polypeptides revealed RFC complex subunits 1-5 (Fig. 12b), which were confirmed by comparing their electrophoretic nobilities with the theoretical masses. Details of mass spectroscopy analysis of the identified subunits and associated protein sequences are shown in the Table 4a and the associated protein sequences are shown in Appendix 8a. Two additional proteins Rad17 and tubulin were also detected. Tubulin is most abundant protein in cells and is not part of the RFC complex. We therefore conclude

32 that this was most likely a contaminant. RFC1 subunits have been shown to be replaced by Rad17 in fission yeast, which forms a pentameric alternative RFC complex derivative Rad17- RFC (Al-Khodairy et al., 1994 and Griffiths et al., 1995), which plays a role in the DNA- damage replication checkpoint response, specifically by loading the 9-1-1 complex onto the DNA. The co-purification of Rad17 in these experiments may suggest that such alternative complexes exist in kinetoplastids as previously (MacNeill, 2014).

To analyse whether we could express and isolate exosome complex subunits in C. fasciculata using the constructed pNUS-PTPcH plasmid, we fused the PTP sequence to the C-terminus of RRP4, which is known to be present in the 11S exosome complex and previously CBP- TAP purified in T.brucei parasites. However, unlike the conventional TAP-RRP4 purification in which only four exosome components in T.brucei were purified (Estevez et al., 2001), we purified all exosome components in C. fasciculata by PTP tagging. Ten subunits were detected after MASCOT peptides mass analysis, which were unambiguously identified as RRP6, EAP1, RRP45, RRP40, RRP4 (Tagged), RRP41B, CSL4, EAP2, RRP41A and EAP4 (Fig. 12c). Details of mass spectroscopy analysis of the identified subunits and associated protein sequences are shown in the Table 4b and the associated protein sequences are shown in Appendix 8b. The observed different sizes of bands corresponding to RRP6 could be due to proteolysis or partial post-translational modification of the protein. We did not find EAP3 subunit in our experiments. Estevez and colleagues also did not TAP purified EAP3 in the cytosolic extracts of T.brucei (Estevez et al., 2001). Perhaps this could be due to the weak interaction of EAP3 with the core exosome complex subunits (EAP3 interacts with the core complex through RRP6) or it was degraded. However, the fact that EAP3 has been isolated in L.tarentolae (Cristodero et al., 2008), further optimizations on extract preparation, and using antisera to confirm this finding should be considered. For wider applicability, further experiments are needed to determine if the purified components are functional. It will be also worthwhile to find out if T.brucei and perhaps Leishmania could also be transformed with this particular construct.

Efficient and high-level heterologous expression of proteins is a crucial step in protein from native sources and is especially useful when the native protein is normally produced in limited amounts or by sources, which are impossible, expensive and/or dangerous to obtain or propagate. We are considering optimising our purification experiments with the anticipation to isolate other novel interacting subunits, which will be validated as potential drug targets in other pathogenic kinetoplastids.

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To conclude, we report the application of tandem affinity purification to C. fasciculata for the first time, demonstrating the effectiveness of the technique by purifying both the intact exosome and replication factor C complexes. Adding tandem affinity purification to the C. fasciculata toolbox significantly enhances the utility of this excellent model system. The present protein expression vector can therefore be used in place of or as an adjunct to other protein expression systems for production of proteins needed for the discovery, evaluation, or production of diagnostics, vaccines, therapeutics or medical treatments of kinetoplastid pathogens.

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3. Chapter 3: Developing a Resazurin-viability assay in Crithidia fasciculata allowing subsequent screening for Anti-Crithidial Compounds from the GSK Open Access Pathogen Boxes

3.1 Introduction

Compounded by massive global food, water shortages and climate change, kinetoplastid diseases continue to have a devastating, long-term impact on both human and animal health and the general welfare in the world, and therefore represent a major global challenge. Although there have been some successes in drug discovery against kinetoplastid diseases for example; the SCYX-7158, and Diamidine series for HAT (Drugs for Neglected Diseases, 2012b); the K777 and triazoles for treating Chagas disease (Barr et al., 2005; Urbina, 2010); the 8-aminoquinoline NPC1161, bis-quinolines series, DB766, rhodacyanine dyes and amiodarone for Leishmaniasis (Richard and Werbovetz, 2010) and the more recent GNF6702 drug candidate against all the three kinetoplastids (Khare et al., 2016), progress in discovering new and effective drugs against the three pathogenic kinetoplatids has been very slow, mostly due to reasons previously discussed (see Chapter 1). One approach in accelerating the discovery of novel leads for the treatment of pathogenic kinetoplastids has been the use of simple, robust and inexpensive cell-based systems to serve as tools for high throughput screening (HTS) of large sets of chemical libraries. However, very few reports are available on HTS assays for the kinetoplastids. A non-human infective kinetoplastid parasite C. fasciculata could enhance HTS and speed up the process of searching for new drugs to treat the diseases caused by the pathogenic kinetoplastids. As previously discussed, C. fasciculata parasites are lower non-humans infective trypanosomatids, which can be handled in a standard laboratory without specific biosafety issues. The parasites can be easily and quickly grown to high densities in less expensive liquid media or fully defined serum-free media and therefore could shortern the long turn around time associated with the pathogenic kinetoplastids screening assays. In this chapter, we report the development and the application of a resazurin reduction--- C. fasciculata cell assay for primary screening and predicting compounds with potential activities against the pathogenic kinetoplastids. In particular, we utilized the developed assay to query the Open Access Chemical boxes for compounds with inhibitory activities against C. fasciculata parasites, which will be followed up against the actual pathogenic kinetoplastids.

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3.1.1 Open access chemical boxes Some few decades ago, an innovative collaboration model for research and development for neglected diseases emerged in the form of public-private partnerships (PPPs), which later came to be known as product development partnerships (PDPs). A notable example of such PDPs are the Medicines for Malaria Venture (MMV) and the Glaxo Smith Kline (GSK) Tres Cantos which were formed with the aim of catalysing the discovery, development and the delivery of new medicines against tropical diseases. Almost seven million compounds have been tested in phenotypic assays against malaria over the last decade, which has resulted in a solid pipeline of new preclinical and clinical candidates (Preston et al., 2016). Moreover, an open science initiative has made many of these structures available online and a collection of 400 key malaria phenotypic ‘hits’, called the ‘Malaria Box’, was launched in 2013. Building on this model, in December 2015, MMV took this a stage further with an initiative to stimulate the drug discovery for neglected parasitic diseases by introducing a Pathogen box. The ‘Pathogen Box’ (www. Pathogen box.org), contains 400 diverse drug-like molecules, which is provided at no cost to research groups. Each of the 400 compounds in the ‘Pathogen Box’ has confirmed activity against one or more key pathogens that cause some of the most socioeconomically important diseases worldwide such as tuberculosis, malaria, sleeping sickness, leishmaniasis, schistosomiasis, hookworm disease, toxoplasmosis and cryptosporidiosis. All the 400 compounds were tested for cytotoxicity with compounds included in the library being at least 5-fold more selective for the pathogen than its mammalian host. Between October 2012 and May 2014, a diverse set of 1.8 million compounds were screened by the GSK Tres Cantos against the three pathogenic kinetoplastids i.e. L. donovani, T. cruzi and T. brucei (Peña et al., 2016). Secondary confirmatory and orthogonal intracellular anti- parasitic assays were conducted, and the potential for non-specific cytotoxicity determined. From this high through put screening, three anti-kinetoplastid chemical boxes were assembled. The selection of representative chemical boxes for the three kinetoplastids started from the most potent, specific, and non-cytotoxic compounds in the dose–response outputs of each screen after having filtered for lead-like properties as described in Pena et al. (2016). In order to generate representative boxes with high chemical diversity and potency, compounds were clustered initially by similarity using a complete-linkage algorithm (Leach et al., 2007) and a threshold of 0.55. Secondly, they were sorted by decreasing potency (i.e. pIC50). The hypothetical biological target space covered by these

36 diversity sets was also investigated through bioinformatics methodologies. The analysis suggested that most of the compounds are new chemical entities with potential novel mechanisms of action that have not been previously exploited against these parasites. Clusters of compound were represented by only two members in the final ranked boxes. The final boxes contained 592 compound entries; 192 were active against L. donovani (Leish-Box), 222 against T. cruzi (Chagas-Box) and 192 against T. brucei (HAT- Box). The three anti-kinetoplastid chemical boxes showed little overlap, pointing to specific mechanisms of growth inhibition or structural divergence across molecular targets in each parasite. Three compounds were in both the Leish-Box and Chagas-Box, nine in both the Chagas-Box and HAT-Box and one compound was present in all three chemical boxes (Peña et al., 2016). Both the Pathogen and the GSK kineto boxes containing compounds are provided free to researchers upon request and the data of all these chemical boxes is publically available with the aim of facilitating and stimulating the drug discovery for these diseases. We therefore took advantage of these boxes to identify compounds with anticrithidial activitivies that will be followed up against the actual pathogenic kinetoplastids.

3.1.2 High throughput phenotypic screening assays Different assays have been developed and investigated to identify new active starting points for drug development against kinetoplastid pathogens. Most of these assays are based on the selection of compound collections and evaluating them in either target-based or phenotypic (whole cell) screening (Pink et al., 2005). Most of the new drug candidates that were approved by the FDA between 1999 and 2008 were identified through phenotypic screening (37% versus 23% discovered by target-based approaches) (Lee et al., 2012). However, the target-based approaches have out numbered phenotypic screening and the success of phenotypic screening has been underrated.

Unlike target-based screens, which rely upon known therapeutic pathways, phenotypic screening has the advantage of identifying new other targets. A compound may affect two or more proteins or pathways in the organism, which would not be identified in a high- throughput target-based screen. Unfortunately, lack of membrane permeability can lead to inactivity being reported for a particular compound that initially demonstrated target activity when assessed in a phenotypic screen. This can be a double-edged sword as a target specific molecule with inactivity in a phenotypic screen would be lost. However, given the costs and

37 frequent failure to suitably optimize and progress molecules for drug discovery, this loss is usually considered acceptable in phenotypic screens (Sykes and Avery, 2013).

In the process of searching for new drugs against kinetoplastid diseases in which very few validated targets exist, a non-reductionist approach such as the phenotypic screening therefore holds significant advantages. Phenotypic screening is considered a cost-effective method of identifying activities of unknown compounds and provide a wider view of the activity that can be hitting either single or multiple targets (De Muylder et al., 2011). Reliable and reproducible phenotypic assays are of great benefits to kinetoplastids drug discovery where assay cost becomes an issue because of the low funding schemes dedicated to kinetoplastid diseases research and the lack of interest in these diseases by large pharmaceutical companies. Recent developments in various platforms for phenotypic screening especially the high throughput screening (HTS) assays has opened new doors of investigation and has allowed the evaluation of significantly larger compound collections (10,000 to several million) representative of a much broader chemical diversity such as those provided by the MMV and GSK Tres Cantos. A number of such whole cell phenotypic screening assays have used succesfully in identifying some novel antikinetoplastid candidates for example; the hydrazine CA272 and a quinolone derivative CH872 for leishmaniasis (Siqueira-Neto et al., 2010), azole antifungals---ianoconazole, bifonazole, and oxiconazole nitrate for T. cruzi, the five new scaffolds--phenylthiazol-4- ylethylamide, phenoxymethylbenzamide, 6-aryl-3-aminopyrazine-2-carboxamide, pyrido- isoxazol-2-ylanilide and aminoethyl benzoylarylguanidine (Sykes and Avery,2013) and the quinolones (Hiltensperger et al., 2012; Fotie et al., 2010) for T. brucei.

3.1.3 Resazurin-reduction cell-based HTS assay The establishment of a simple in vitro cell culture systems for axenic growth of kinetoplastids has led to the exploration of a various whole cell assay formats to serve as tools for HTS, with the aim of assessing large sets of chemical libraries and prioritize those for further synthesis of analogues in hit-to-lead and lead optimisation phases of the drug discovery process (Muskavitch et al., 2008). For evaluating the cell viability following exposure to test compounds, the resazurin (Alamar Blue™) (Räz et al., 1997; Sykes and Avery, 2009) and Cell-Titer-Glo™ luminescent cell viability assay (Mackey et al., 2006) methods have emerged as those most amenable to HTS because of their high signal-to-background ratio and reproducibility. Generally, these assays are performed in "automation-friendly" microtiter plates with either a 96, 384 or 1536 well format.

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The resazurin-based assay is preferred due to its simplicity, low cost, lack of radioactive materials and non-toxic and transferability to the field if necessary. It has been extensively used for screening of drug susceptibility in whole cell cultures of trypanosomes and other cell lines for so many years (Bowling et al., 2012; Sykes and Avery, 2009, Räz et al., 1997; Shimony and Jaffe, 2008; Nare et al., 2010 ), Leishmania ( Fumarola et al., 2004b) human cells (Ahmed et al., 1994; O’Brien et al., 2000), fungi (Tiballi et al., 1995), bacteria (Baker and Tenover, 1996; Franzblau et al., 1998). Resazurin is an active ingredient of alamar blue. Resazurin dye is a water-soluble, non-toxic, permeable through cell membranes and is stable in culture medium. It is highly dichromatic based on Kreft's dichromaticity index (Kreft and Kreft, 2009). The dye acts as an intermediate electron acceptor in the electron transport chain without interference of the normal transfer of electrons (Page et al., 1993). The oxidation-reduction potential of resazurin is +380 mV at pH 7.0, 25 °C. It can therefore be reduced by NADPH (Eo = 320 mV), FADH

(Eo = 220 mV), FMNH (Eo = 210 mV), NADH (Eo = 320 mV), as well as the cytochromes

(Eo = 290 mV to +80 mV) (Rampersad, 2012). Resazurin can be converted from its oxidized, non-fluorescent, blue colour to the reduced, highly fluorescent, pink coloured resorufin that can further be reduced to nonfluorescent uncoloured dihydroresorufin (Page et al., 1993) (Fig. 13). Mitochondrial reductases and other enzymes such as the diaphorases (EC 1.8.1.4, dihydrolipoamine dehydrogenase) (Matsumoto et al., 1990), NAD (P) H: quinone oxidoreductase (EC 1.6.99.2) (Belinsky and Jaiswal, 1993) and flavin reductase (EC 1.6.99.1) (Chikuba et al., 1994) located in the cytoplasm and the mitochondria may be able to reduce resazurin. Therefore, resazurin reduction may signify an impairment of cellular metabolism and is not necessarily specific to interruption of electron transport and mitochondrial dysfunction (Mood and Mommsen, 2005). This change from oxidized to reduced state allows flexibility of detection where measurements can be quantitative as colorimetric and/or fluorometric readings (the latter being more sensitive) or qualitative as a visible change in colour indicating presence or absence of viable cells. These properties of resazurin have been exploited to provide a quantitative measurement of parasite proliferation and viability to identify a variety of inhibitor compounds.

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Figure 13. Schematic of Resazurin reduction reaction. Reducing environment in viable cells continuously converts the essentially non-fluorescent resazurin to a highly fluorescent resorufin, increasing the overall fluorescence and colour of the media surrounding cells. The resorufin is further reduced to uncoloured and no fluorescent dihydroresorufin.

Indeed, utilizing these robust and reliable assays with convenient organisms like C fasciculata and taking advantage of the available open access chemical boxes in drug discovery screening cascades could play a very crucial role in speeding up the process of searching for new drugs against kinetoplastids.

3.2 Materials and methods 3.2.1 Parasites and cell culture The C. fasciculata promastigotes clone HS6 was used in all assays and parasites were grown at 27oC with a gentle agitation in axenic serum-free defined culture media containing Yeast extract, Tryptone, Sucrose, Triethanolamine, Tween 80 and supplemented with haemin as described in Appendix 2. All chemicals and reagents used in the experiments were of highest grade and were purchased from Sigma-Aldrich. The parasites were sub-cultured every 2-3 days to ensure log growth phase for subsequent experiments. 3.2.2 Compounds libraries Chemical boxes were kindly provided by MMV and GKS Tres cartos following a request. The Pathogen Boxes contained 400 chemicals representing compounds that were active against one or more of 12 distinct pathogens (http://www.pathogenbox.org/about-pathogen- box/supportinginformation). Individual compounds had only been tested to confirm activity against the pathogen for which the compounds were first reported to be active, and have not been tested against the other pathogens represented in the Pathogen Box. All compounds have been tested for cytotoxicity; typically, they were five-fold less potent against a human fibroblast cell line (MRC-5) than the pathogen (www. pathogenbox.org/about-pathogen- box/supporting-information).

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The three GKS kineto chemical boxes (Leish-box), (Chagas-box) and HAT box) with each box containing ~200 compounds assembled by Pena and his colleagues (Pena et al., 2016) as previously discussed, were donated by GSK Tres-Cartos. These compounds included details on the pathogen against which the compound was shown activity, their cytotoxicity as well as other useful data such compound ID, batch ID, trivial name, molecular weight, salt, and cLogP. More information about these compounds can also be accessed online via ChEMBL-NTD (https://www.ebi.ac.uk/chemblntd).

Both the MMV and the GSK compounds were supplied in 96-well plates, containing 10 µL of 10 mM dimethyl sulfoxide (DMSO) solution of each compound. Each compound was then diluted with PBS to a working concentration of 2.5 mM (DMSO 25%) working and aliquoted into multiple plates. The compounds were stored at -80°C and thawed at room temperature prior to use. Each of the 400 compounds was screened in quadruplicate at a concentration of 100 µM (DMSO 0.5% final concentration) in 96-well plates. Subsequent pure compounds were repurchased from Sigma-Aldrich for structure-activity relationships (SARs). Similary, these compounds were prepared in stock concentration of 10 mM with DMSO and screened at 100 µM (DMSO 0.5% final concentration). The Alamar Blue solution was prepared by dissolving 12.5 mg Resazurin sodium salt (Sigma-Aldrich) in 100 ml of Phosphate Buffered Saline (PBS). The solution was then sterilized by filtration with a 0.22 µm Millipore Express PLUS membrane filter and used immediately. All fluorescence measurements in this study were performed with the Spectra Max Gemini XPS Microplate reader (Gemini XPS, Molecular devices) with excitation wavelength of 530 nm and 560 nm. 3.2.3 Resazurin-reduction C. fasciculata cell-based assay optimization Although resazurin-reduction assay has been extensively used for screening drug susceptibility of various cell types, none has tempted to apply this assay in Crithidia. Therefore, a number of conditions such as; the growth kinetics of cells, maximum cell densities, the incubation period, the resazurin concentration and the DMSO concentrations had to be considered and optimised for the resazurin-reduction assay to work as a screening tool in this system.

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3.2.3.1 The multiplicative kinetics of C. fasciculata Cell densities of three replicate cultures starting at 1x103, 1x104 and 3x104 cells/ml were microscopically monitored and counted using a haemocytometer at 24 hour intervals, over 5 days. A growth curve was plotted to estimate the doubling time and the maximum number of cells attainable in a 25-cm2 before stationary phase and possible cell death occur. 3.2.3.2 Determining the effect of incubation period and the volume of resazurin on fluorescence development In order to determine the fluorescent development at different volumes of the dye and the incubation period, C. fasciculata choanoamastigotes (5x104cells/ml) were incubated in the presence of various resazurin dye volumes (5, 10, 15 and 20 µl) and monitored after every 1 hour for a period of 4 hours. The experiment was performed twice and the results were averaged over eight replicate wells. 3.2.3.3 Determining the relationship between cell density and the Resazurin fluorescence To determine the relationship between cell density and the fluorescence signal, the parasites in the logarithmic phase of a stock suspension of 80x106cells /ml were serially diluted (100 µl) into 96-well plates followed by addition of 10 µl of resazurin. Plates were incubated at 27°C and fluorescence measured after every 1 hour for a period of 4 hours. The experiment was performed twice and the results were averaged over eight replicate wells. 3.2.3.4 Determining the effect of DMSO concentrations on the assay signal A 90µl of medium containing C. fasciculata choanoamastigotes (5x103cells/ml) was inoculated into a 96-well plate and incubated for 24-hours. Ten microliters of various (0.5-9% final) concentrations of DMSO diluted in the medium were then added to the plates and further incubated for 24 hours. The experiments were performed twice and the results were averaged over eight replicate wells. 3.2.4 Compound sensitivity assays 3.2.4.1 Primary screening assays C. fasciculata choanoamastigotes in the log phase of growth were diluted 1:20 in the growth media, and 20µl was counted using a hemocytometer. For anti-crithidial activity, compounds were added to the test plates with medium containing the parasites (density: 5x103cells/ml) to achieve a final compound and DMSO concentration of 100 µM and 0.5%, respectively. The controls on each plate included wells containing growth media--0.5% DMSO without cells (Positive control) and growth media--0.5% DMSO with cells only (Negative control). The activities of test compounds were normalized against controls from the same plate according

42 to the following formula: Activity (%) = [1 − (FCpd − FPos) / (FNeg − FPos)] ×100, where FCpd corresponds to the emitted fluorescent signal expressed in arbitrary fluorescence units for the test compound; and FNeg and FPos correspond to the mean fluorescent signal of the negative and the positive control wells, respectively. For estimation of the hit confirmation rate, compounds were considered “confirmed” when the normalized anti-parasitic activity was equal to or greater than 80% (≥80%) at 100 µM concentration. 3.2.4.2 Dose-response assessments of active compounds Compounds which showed ≥80% inhibition when tested at 100 µM concentration in at least one biological replicate were re-tested in 10-point dose response, two-fold dilution experiments starting at various compound concentrations with the parasites seeding density of 5x103cells/ml. Wells containing the 0.5% DMSO growth media with no cells and 0.5% DMSO growth media with cells but no drug served as 100% inhibition and 100% growth controls, respectively. Pentamidine and suramin were used as reference compounds. A 10 µl of Resazurin® was added after 44 hours incubation and fluorescence development was determined after a total drug exposure time of 48 hours. The obtained fluorescence data was analysed with the graphic data analysis software “GraFit” which calculated EC50 values by linear regression from the sigmoidal dose inhibition curves.

Compounds which did and did not yield an EC50 value within the confines of the analysis parameters were simply expressed as the “true active” and “false active” compounds, respectively. A few compounds of interests, which had comparably low EC50 values were cherry picked from the top ten lists, purchased from commercial sources (Sigma-Aldrich) and screened to finally confirm their activities.

EC50 was defined as the amount of a compound required to decrease the C. fasciculata viability by 50% compared to those grown in the absence of the test compound. All experiments were performed twice, with each drug concentration in quadruplet. For standardisation, of EC50 values were converted to pIC50 using a converter found at www.sanjeevslab.org/tools.html. According to the FDA, pIC50 is the recommended way of measuring/reporting the effectiveness of a substance in inhibiting a specific biological or biochemical function. The pIC50 represents the concentration of a drug that is required for 50% inhibition in vitrowhile EC50 mainly represents the plasma concentration required for obtaining 50% of a maximum effect in vivo.

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3.3 Results and Discussion 3.3.1 Resazurin-reduction C. fasciculata cell-based assay optimization In order to determine the maximum cell numbers that could be used for developing the screening assay, the growth kinetics of C. fasciculata parasites growing in our formulated serum free medium was analysed using the growth curve as shown (Fig.14). The parasites grew quite robustly under axenic conditions in vitro and reached the stationary phase after 3 days. Similar C. fasciculata choanoamastigotes growth kinetics in in vitro culture systems have been reported elsewhere (Calderón-Arguedas et al., 2006 and Scolaro et al., 2005). An average generation time was determined according to Popp and Lattorff (2011) equations and gave an estimation of approximately 4.5 hours. The doubling time observed is shorter than the doubling time (6.8 hours) reported for T. brucei brucei blood foams when grown in HMI- 9 supplemented with 10% fetal calf serum (Skyes and Avery, 2009) and 7 hours for Leishmania species (personally communicated by Menzies S, Terry Smith’s laboratory).

Figure 14. Growth curve of C. fasciculata choanoamastigotes. Cultures starting at 1000, 10000 and 30000 cells/ml were microscopically monitored and counted using a haemocytometer at 24 hours intervals for 5 days. Average counts were obtained from two biological replicates and the experiment was repeated twice.

A linear relationship was observed between the incubation time and the fluorescent development of Resazurin reduction (Figure 15). However, low dye concentrations gave relatively higher fluorescent signal as compared to high concentrations. Skyes and Avery (2009) and Raz et al. (1997) also observed a similar independent relationship between

44 resazurin concentration and the fluorescent signal in their assay development. Obviously, this is due to quick reduction of small volumes of the dye by the cells or perhaps high concentrations of resazurin salts had inhibitory effect on cell growth and metabolism. For higher cell inoculums, the fluorescent signal easily reached saturation within some few minutes when a 5% (5 µl) dye concentration was used but we were able to get strong fluorescent signal after 4 hours incubation with a 10% (10 µl) resazurin. We therefore considered using the 10% resazurin as an ideal dye concentration for all of our assays. The fluorenscence signal correlated with cell numbers (Fig.16). However, for high cell densities such as 80x106/ml and 40x106/ml the fluorescence signal reached saturation after 1 hour and 2 hours incubation, respectively. We were able to obtain a very strong signal with 20x106 cells/ml giving the best maximum fluorescence to background signal ratio of 9:1. The reported signal to background ratio (S/B) is much higher than the 3:1 obtained on T. b. gambiense and but lower than on T. b. rhodesience (15:1) in similar assays (Raz et al., 1997). These differences could be attributed to variations in the dehydrogenase activity responsible for metabolizing resazurin or reduced uptake of the dye substrate among the parasites. Differences in the fluorescence analysers, concentrations of the dye used as well as the composition of media used to culture these parasites could potentially also account for some of the variations observed. Future studies should however, aim to accurately determine the linearity between the fluorenscence signal and the cell density. For example, carrying out the experiment at much lower parasite density than 80 x106 cells/ml or perharps using direct transfer (inoculation) of known cell densities (starting at very lower densities) from the culture to the wells according to Sykes and Every (2009) and Raz et al (1997).

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Figure 15. The effect of incubation period and the volume of resazurin on fluorescence development. The fluorescence was monitored by incubating C. fasciculata choanoamastigotes (5x104cells/ml) in the presence of various resazurin volumes (5, 10, 15 and 20 µl) at 1 hour interval for 4 hours period. All experiments were performed twice and average signals plotted.

Figure 16. Relationship between the C. fasciculata choanoamastigotes density and resazurin fluorescent signal. Serial two-fold dilutions of parasites starting at 80 x106 cells/ml were prepared in 100 µl followed by addition of resazurin and fluorescence measurement to determine parasite viability. All experiments were performed twice and average signals plotted.

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Since compounds library collections were diluted in DMSO, which is known to be toxic to various cells, we exposed the parasites to various DMSO concentrations to determine wether it had any effect on the cells viability. The C. fasciculata parasites were able to tolerate maximum DMSO concentrations of up to 0.5% with no significant decrease in fluorescent signal (Fig. 17). This DMSO sensitivity is a bit higher than 0.42% reported for blood forms T. b. brucei by Skyes and Avery (2009) but comparably lower than that reported on blood stream forms of T. brucei and T.brucei congolense (Merschjohann and Steverding, 2006). Since C. fasciculata parasites grows almost everywhere including the harsh environments and unprotected from the UV from the sun, we expected the parasites to more resistant to DMSO than the blood stream forms trypanosomes. The fact that the blood stream forms trypanosomes also endure a harsh host immune system should also not be ruled out as a possibility of their advantage to with stand such toxic concentrations of DMSO. Nevertheless, one other possible factor that may have resulted to such different observations of DMSO sensitivities might be due to the nature of the medium used in each of the protocols. Different culture medium may have different constitutes which may positively or negatively react with the DMSO effecting the viability and consequently the doubling times of the parasites. Moreover, the use of water to dilute compounds have been observed to possess significant effects on the cell viability and EC50 value of the compounds possibly due to osmotic effect of water on cells and changes in buffering capacity of the medium as reported somewhere (Sykes and Avery, 2009).

After optimizing conditions such as cell concentrations, incubation times, resazurin concentration and the DMSO concentration, the assay performance and its capabilities to discriminate the activities of different compounds was determined by calculating the Z` factor, a statistical parameter for use in evaluation and validation of high throughput screening assays (Zang et al.,1999). Statistically, the assay performed well according to the Z` factor criteria (cut off=0.5, but closest to 1 as possible) by obtaining an average Z` factor of 0.7 (a maximum plating cell density of 5x103cells/ml, 48 hours incubation, 10% v/v resazurin and max 0.5% DMSO). The distribution Z` factor in a total of 100 randomly selected plates (Fig.18) also confirmed that the assay was able to discriminate compounds with different levels of inhibition in C. fasciculata viability assay during the screening process.

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Figure 17. Dimethyl sulfoxide (DMSO) concentration and fluorescent signal. A cell density of 5x103cells/ml was incubated for 48 hr in various DMSO concentrations and resazurin (10%) florescent signal measured. The experiment was performed twice with signal at each DMSO dose averaged from quadruplicate samples

Figure 18. Distribution of Z` factors in a total of 100 plates randomly selected from the MMV (25 plates) and; GSK T. brucei (25 plates), T. cruzi (25 plates) and Leishmania (25 plates) boxes.

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3.3.2 Screening GSK pathogen boxes for anti-crithidial compounds with a resazurin-reduction assay.

Utilizing the conditions established during optimization, the Resazurin reduction--C. fasciculata assay was used to screen for anti-crithidial compounds in the Open Access Chemical boxes (MMV pathogen box and GKS chemical boxes) to identify a workable number of potential compounds for follow up testing against the actual pathogenic kinetoplastids. Using an inhibition cut-off of ≥80%, the primary screening of MMV pathogen box led to the identification of 91 (23%) compounds with inhibitory activities against C. fasciculate (Fig. 19) (Appendix 9). The potency of the 91 compounds was further evaluated through dose- response experiments to determine their IC50 values. This led into the identification of 72 (79%) true active compounds and 19 false active compounds thus representing 18% hit rate.

Figure 19. The workflow used to identify and progress hits of the open access pathogen boxes including key criteria considered in the decision-making process. In this case, all the compounds that had inhibition of ≥80% were progressed to dose-response experiments to determine their IC50 values. True active compounds are those that had a ≥80% inhibition and showed an IC50 in dose-response experiments.

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Ten compounds (top ten) were then cherry picked from the 72 true active compounds in the MMV boxes and the profiles of these top ten hits are shown in Table 5. Strikingly, three compounds (1, 6 and 9) shared a pyrimidin-4-amine chemical fragment. Pyrimidin-4-amine derivatives are well-known kinase and cytochrome P450s inhibitors in various organisms (Pena et al., 2016 and Gunatilleke et al., 2012). One compound (5) had a quinazoline-2, 4-diamine series well referenced in the literature as inhibitors of folate synthase pathways in Leishmania, Trypanosoma and Plasmodium (Pez et al., 2003; Khabnadideh et al., 2005; Muller and Hyde, 2013).

From the GSK boxes, the primary screening of a T. brucei box identified a total of 66 compounds (35%) with inhibitory activities against C. fasciculata (Appendix 10), of which only 42 (64%) were true active representing a hit rate of 22%. Screening the T. cruzi and Leishmania boxes identified 101(46%) and 122(67%) compounds with inhibitory activities against C. fasciculata (see Appendix 11 and 12, respectively), of which 68 (67%) and 89(73%) were true active, respectively (Fig. 19), representing the hit of 31% and 49% for T. cruzi and Leishmania box, respectively. The enriched hits rates observed among the GSK boxes suggest the commonality of the targets shared between C. fasciculata and the T. brucei, T. cruzi, and to the large extent the leishmania species. Since all the GSK compounds were previously shown to be active against each of the respective kinetoplastids in vitro (Pena et al., 2016), this might suggest that the hits have favourable properties to reach their active biological targets shared between C. fasciculata and the pathogenic kinetoplastids in these assays.

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Compound pIC50 cLogP Compound name Mwt Structure of the ID compound 1 MMV0 7.4 ± 3.55 N-cyclohexyl-6-cyclopropyl-2- 294.40 21013 0.004 (pyridin-2-yl)pyrimidin-4- amine

2 MMV2 7.7 ± -0.27 1-(3-methoxyphenyl)-5- 254.27 72144 0.03 (methylsulfonyl)-1H-tetrazole

3 MMV6 8 ± 0.001 2.81 (S)-2-nitro-6-((4- 359.26 88755 (trifluoromethoxy)benzyl)oxy)- 6,7-dihydro-5H-imidazo[2,1- b][1,3]oxazine 4 MMV6 8 ± 0.003 4.26 N-(4-(trifluoromethyl)phenyl)- 466.42 89243 N-(1-(5- (trifluoromethyl)pyridin-2- yl)piperidin-4-yl)pyridin-3- amine 5 MMV6 8 ± 0.005 2.31 5-chloro-6-(((2,5- 359.81 75968 dimethoxyphenyl)amino)methyl )quinazoline-2,4-diamine 6 MMV6 6.1± 0.01 3.93 5-chloro-N,6-dimethyl-N- 338.84 58988 phenethyl-2-(pyridin-2- yl)pyrimidin-4-amine

7 MMV5 6.3 ± 0.1 -0.31 3-((methylsulfonyl)methyl)-2H- 239.25 53002 benzo[b][1,4]oxazin-2-one

8 MMV6 7±0.04 5.04 2-methyl-6-nitro-2-((4-(4-(4- 534.48 88262 (trifluoromethoxy)phenoxy)pipe ridin-1-yl)phenoxy)methyl)-2,3- dihydroimidazo[2,1-b]oxazole 9 MMV6 6.1±0.02 3.12 1-(2-(1- 464.58 88470 (methylsulfonyl)piperidin-4- yl)ethyl)-3-(naphthalen-1- ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine

10 MMV6 6.1±0.01 2.71 2-(((1-propyl-1H- 281.35 76445 benzo[d]imidazol-2- yl)methyl)amino)phenol

Table 5. Profiles of the top ten hits identified from screening of the MMV pathogen box. Compounds with pyrimidin-4-amine scaffold are highlighted in red.The cytotoxicity of all these compounds are at least 5-fold more selective for the pathogens than their mammalian hosts. The cLogP, cytotoxicity data and other relevant information of these compounds are found at (http://www.pathogenbox.org/about-pathogen-box/supportinginformation).

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Ten compounds (top ten) were then cherry picked from the list of the true active compounds in each of the T. brucei, T. cruzi and Leishmania box and the profiles of the top ten hits are shown in Table 6, 7 and 8, respectively.

From the T. brucei GSK box (Table 6), two compounds (4 and 8) also had a pyridine-4- amine chemical fragment while three compounds (1, 5 and 10) shared a 5--2-yl fragment and the other compounds had distinct structures. The nitro-substituted aryl group derivatives are substrates of type I nitroreductases of various parasites which are metabolized into toxic nitrile products harmful to the parasites (Hall et al., 2011). Nevertheless, the 5- nitrofuran-2-yl derivatives also inhibitors of Myco-bacterium tuberculosis H37RV (Doreswamy and Chanabasayya, 2013). The fact that there are no mammalian homologues to the 5-nitrofuran-2-yl targets place their derivatives as potential drug candidates against various pathogens. The increasing interest in treating kinetoplastids diseases with nitro drugs such as nifurtimox and benznidazole (Patterson and Wyllie, 2014), and the nitro-aromatic chemicals present in these sets calls for further studies on their substrate selectivities.

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Compound pIC50 Cytotoxi cLogP Compound name Mwt Structure of the ID city compound

1 TCMDC 5.5±0.3 <4.0 3.422 2-(4-chlorophenyl)- 291.65 -143074 5-(5-nitrofuran-2- yl)-2H-tetrazole

2 TCMDC 5.4±0.0 <4.0 1.21 2-(3-(4- 281.22 -143457 1 (difluoromethoxy)-3- 1 methoxyphenyl)- 1,2,4-oxadiazol-5- yl)acetonitrile

3 TCMDC 5.4±0.2 4.7 1.881 4-bromo-2- 227.01 -143609 nitrobenzonitrile 8

4 TCMDC 5.3±0.0 4.5 2.772 N-(5-cyclopropyl- 284.36 -143363 3 1H-pyrazol-3-yl)-6- 7 methyl-2- (pyrrolidin-1- yl)pyrimidin-4- amine 5 TCMDC 5.3±0.1 4.8 2.654 (2- 295.31 -143112 (diethylamino)thiaz 9 ol-5-yl)(5- nitrofuran-2- yl)methanone 6 TCMDC 5.1±0.5 4.5 3.74 5-methyl-7-(2,4,5- 329.57 -143316 trichlorophenoxy)- 5 [1,2,4]triazolo[1,5- a]pyrimidine

7 TCMDC 5.0±1.0 <4.0 4.29 3-chloro-N-(5- 307.13 -143172 chlorobenzo[d]oxazol 2 -2-yl)benzamide

8 TCMDC 5.0±0.7 <4.0 2.383 N-(4,5-dimethyl-1H- 227.26 -143460 pyrazol-3-yl)-1H- 5 pyrrolo[2,3- b]pyridin-4-amine

9 TCMDC 5.0±0.0 <4.0 1.358 4-(benzylcarbamoyl)- 357.36 -143079 4 1,2-phenylene 1 bis(methylcarbamate)

10 TCMDC 5.0±0.3 <4.0 1.212 3-(5-(5-nitrofuran-2- 258.19 -143073 yl)-2H-tetrazol-2- 8 yl)pyridine

Table 6. Profile of ten top hit compounds identified in the GSK T.brucei box. Compounds with pyrimidin-4-amine scaffold are highlighted in red and those with 5-nitrofuran-2-yl scaffold are in blue. Data on cytotoxicity and cLogP and other information about these compounds is found on (https://www.ebi.ac.uk/chemblntd).

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No compounds with common scaffolds were identified in the T. cruzi box (Table 7). However, of particular interest, we identified one compound (3) with a quinolin-8-ol chemical scaffold previously reported for their antifungal properties but of which their mode of action is not yet known (Musiol et al., 2006). We also identified one compound (8) in the T. cruzi box, which shared a common 2, 2, 2-trifluoroacetate moiety with two compounds (2 and 10) in Leishmania box (Tables 7 and 8). The Leishmania box also revealed two more compounds (1 and 2) with pyrimidin-4-amine based structure (Table 8). The increased number of active compounds sharing a pyrimidin-4- amine structural class identified in both the MMV and GSK boxes strongly suggest the need for follow up testing of their analogues against the pathogenic kinetoplastids.

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Compound pIC50 Cytoto cLogP Compound name Mwt Structure of the ID xicity compound 1 TCMD 5.9±0.0 <4.0 -0.242 5-((allyloxy)methyl)-2- 226.18 C- 5 nitro-5,6- 9 143149 dihydrooxazolo[3,2- b][1,2,4]triazole 2 TCMD 5.6±0.1 <4 0.432 1-(1-methyl-3-nitro-1H- 211.22 C- 1,2,4-triazol-5- 1 143088 yl)piperidine

3 TCMD 5.7±0.0 4.7 2.799 2-methyl-7-((pyridin-2- 342.39 C- 1 ylamino)(pyridin-3- 4 143308 yl)methyl)quinolin-8-ol

4 TCMD 5.8±0.0 <4.0 3.005 ((1S,2S)-2-((bis(pyridin- 387.47 C- 3 3- 4 143593 ylmethyl)amino)methyl) cyclopropyl)(3- methoxyphenyl)methano ne 5 TCMD 5.7±0.0 4.3 3.947 1-(4-(4-bromo-2- 419.65 C- 7 chlorophenoxy)butyl)- 5 143590 1H-imidazole oxalate

6 TCMD 5.6±0.0 4.7 2.666 6-ethyl-7- 231.29 C- 1 propylpyrido[2,3- 7 143606 d]pyrimidine-2,4- diamine

7 TCMD 6.5±0.0 4.3 4.109 2-(4-(4-((4- 423.95 C- 2 chlorophenyl)sulfonyl)pi 7 143622 perazin-1-yl)phenoxy)- N,N-dimethylethan-1- amine 8 TCMD 6.2±0.0 <4.0 3.173 (3aS,7aS)-2-(2- 411.393 C- 5 fluorophenyl)-5-(pyridin- 143422 3-yl)octahydro-1H- pyrrolo[3,4-c]pyridine 2,2,2-trifluoroacetate 9 TCMD 6.3±0.0 4.4 1.913 N-(cyclohexylmethyl)- 225.311 C- 03 1,2,3-thiadiazole-5- 143612 carboxamide

10 TCMD 5.7±0.0 <4.0 2.525 (2-chloro-4-(pyrrolidin-1- 321.845 C- 1 yl)phenyl)(4-methyl-1,4- 143127 diazepan-1-yl)methanone

Table 7. Profile of ten top hit compounds identified in the GSK T. cruzi box. Compound with quinolin-8-ol and 2, 2, 2-trifluoroacetate scaffolds are highlighted in green and yellow, respectively. Data on cytotoxicity, cLogP and other information about these compounds is found on (https://www.ebi.ac.uk/chemblntd).

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Compound pIC50 Cytoto cLogP Compound name Mwt Compound structure ID xicity

1 TCMD 6.9±0.0 4.3 3.7 N-(3-methoxyphenyl)- 292.3 C- 1 6-methyl-2-(pyridin-2- 43 143621 yl)pyrimidin-4-amine

2 TCMD 6.8±0.0 4.8 3.597 6-cyclopropyl-2-(1- 433.4 C- 03 methyl-1H-imidazol-2- 37 143487 yl)-N-(2methyl benzyl)pyrimidin-4- amine2,2,2- trifluoroacetate 3 TCMD 6.0±0.2 <4.0 3.648 N-(6 ethyl 368.4 C- benzo[d]thiazol-2-yl)-4 61 143239 morpholinopicolinamid e 4 TCMD 6.0±0.0 4.0 3.005 ((1S,2S)-2- 387.4 C- 2 ((bis(pyridin-2- 74 143586 ylmethyl)amino)methyl )cyclopropyl)(3methox yphenyl)methanone 5 TCMD 5.9±0.1 <4.0 4.188 5-ethyl-N-(1-phenyl- 297.3 C- 1H-imidazol-2- 81 143375 yl)thiophene-3- carboxamide

6 TCMD 5.6±0.0 <4.0 3.07 2-(((1-butyl-1H- 302.4 C- 6 tetrazol-5- 04 143315 yl)methyl)thio)-4,6- dimethylnicotinonitrile

7 TCMD 5.5±0.2 4.4 3.856 N-(4-(pyridin-2- 335.4 C- yl)thiazol-2-yl)-1,2,3,4- 31 143113 tetrahydronaphthalene- 2-carboxamide 8 TCMD 5.4±0.0 <4.0 3.349 N-benzyl-2-((1-phenyl- 358.3 C- 1 1H-pyrazolo[3,4- 93 143358 b]pyridin-3- yl)oxy)acetamide 9 TCMD 5.5±0.5 <4.0 4.289 N-butyl-4- 338.4 C- isobutyramido-N- 43 143252 phenylbenzamide

10 TCMD 5.5±0.3 5.0 1.55 4-(5-amino-3-phenyl- 1242. C- 1H-pyrazol-1-yl)-6- 549 143218 (pyridin-2-yl)-1,3,5- triazin-2-amine octakis (2,2,2-

trifluoroacetate)

Table 8. Profile of ten top hit compounds identified in the GSK Leish box. Compounds with pyrimidin-4-amine and 2, 2, 2-trifluoroacetate scaffolds are highlighted in red and yellow, respectively. Data on cytotoxicity, cLogP and other information about these compounds is found on (https://www.ebi.ac.uk/chemblntd).

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A total of eight chemical fragments were chosen from the top ten lists of active compounds from the respective chemical boxes and their near neighbors purchased from commercial sources. These fragments were prioritised based on: (i) existing in vitro or in vivo data regarding their potency and efficacy in other applications (ii) evaluation of their chemical structure for drug-like potential and metabolic liabilities; and (iii) the availability of near neighbors for SAR studies. The purchased near neighbors were then analysed in dose- response experiments to confirm their potency. Unfortunately, no significant improvement in the in vitro activities was observed in the pure near neighbour compounds of the chosen fragments. This could be possibly due to the general physicochemical properties of these pure compounds. However, compounds 1, 2 and 4 harbouring an uncumbered 2-pyridinyl moety were observed to be more potent compared to other near neighbors (Table 9).

Name of the compound pIC50 Mwt Structure 1 4,5-dichloro-6-methyl-2-(2- 5.0±0.6 240.09 pyridyl)pyrimidine

2 2-(2-pyridinyl)-6-(trifluoromethyl)-4- 5.3±1.0 241.17 pyrimidinol

3 2-(4,6-Dimethyl-pyrimidin-2-yl)-5- 4.0±0.2 203.24 methyl-2H-pyrazol-3-ylamine

4 5-(isopropylsulfonyl)-2-(2- 5.4±0.7 278.33 pyridyl)pyrimidin-4-amine

5 Ethyl 4-methyl-1,2,3-thiadiazole-5- 4.2±0.7 172.21 carboxylate

6 1,2,3-thiadiazole-4-carboxylic acid 4.1±1.0 130.12

7 Cyclohexanemethyl-amine 4.4±0.4 113.20

8 N-(cyclohexylmethyl)-4-oxo-4H- 3.9±0.6 285.34 chromene-2-carboxamide

Table 9. Profiles and pIC50s of near neighbour compounds based on the more potent compounds in the top ten lists of the chemical boxes. Compound number 8 (N- (cyclohexylmethyl)-4-oxo-4H-chromene-2-carboxamide) have structural similarity to compound number 7(Cyclohexanemethyl-amine) all of them showing comparably low potency.

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The potency of near neighbors harbouring a 2-pyridyl ring provided confidence in the anti- crithidial activities of compounds harbouring this moety. Derivatives of 2-pyridyl are well reported as CYP51 inhibitors, previously shown to enhance the anti-Mycobacterium tuberculosis and anti-trypanosome activity of 2-aminothiazoles (Meissner et al., 2013 and Kaiser et al., 2015). Nevertheless, two pyridinyl derivatives, (S)-(4-chlorophenyl)-1-(4-(4- (trifluoromethyl) phenyl)-piperazin-1-yl) -2-(pyridin-3-yl) ethanone and the N-[4- (trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine have also been identified as promising drug candidates in animal models of Chagas disease(Hargrove et al., 2013). The reasons to the failure to retain potency observed in the other near neighbors might be because of inefficient pin transfer or perharps due to other physicochemical properties of these pure solid compounds. However, as is crucial for any screening approach, our hit definition was set to identify a workable number of compounds for follow-up, and despite the relatively modest sensitivity of our assay, it enabled the identification of compounds with a high likelihood of confirmed activity when cherry-picked for follow-up testing.

In conclusion, we have developed a simple drug screening system with C. fasciculata that can be used to predict compounds with potential activities against the pathogenic kinetoplatids. Using the developed assay, we repurposed the open access chemical boxes for anti-crithidial compounds and we have identified attractive chemical scaffolds, which will be considered in follow up testing against the actual pathogenic kinetoplastids. The utilization of C. fasciculata to predict compounds with potential activities against the pathogenic kinetoplastids could therefore provide a less expensive but easy and faster altenative approach in search of potential drug candidates against these pathogens.

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4. Chapter 4: The effects of ionizing gamma radiation on the kinetoplastid Crithidia fasciculata

4.1 Introduction 4.1.1 Gamma ionizing irradiation and its effects on cells Gamma irradiation is described as electromagnetic radiation of short wavelength emitted by radioactive isotopes with unstable nuclei that break up and decay to reach a more stable form. It has been widely used for sterilization of medical devices, food preservation and processing of tissue allografts and blood components avoiding the need for high temperatures that can damage such products (Hansen and Shaffer, 2001; Kainer et al., 2004; Mendonca et al., 2004; Osterholm and Norgan, 2004). For the past few decades, studies on the effects of ionizing irradiation stress have been an important issue in different areas of interest, from environmental safety and industrial monitoring to aerospace and currently in biology.

The absorption of ionizing radiation by living organisms may directly disrupt atomic structures and produce chemical and biological changes in their cells (Azzam et al., 2012). Radiation may also act indirectly through radiolysis of water, thereby generating reactive chemical species that may damage nucleic acids, proteins and lipids (Eric and Amato, 2006) (Fig. 20).

The combined direct and indirect effects of radiation may initiate series of biochemical and molecular signalling events that may repair the damage or progress into permanent physiological changes that may consequently lead to cell death (Sharma et al., 2012). The oxidative biochemical changes may continue to arise for days and sometimes months after the initial exposure possibly because of continuous generation of reactive oxygen (ROS) and nitrogen (RNS) species in the cells (Petkau, 1987). Surprisingly, these processes occur not only in the irradiated cells but also in their progeny (Spitz et al., 2004; Kryston et al., 2011; Sharma et al., 2012).

The radiation-induced oxidative stress may spread from targeted cells to non-targeted bystander cells through intercellular communication mechanisms (Azzam et al., 2012; Seymour and Mothersill, 2004; Prise and O'Sullivan 2009) (Fig. 21). The progeny of these bystander cells also experience changes in their oxidative metabolism and may exhibit a wide range of oxidative damages such as protein carboxylation, lipid peroxidation, and enhanced rates of spontaneous gene mutations and neoplastic transformations (Buonanno et al., 2011).

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Figure 20. Direct and indirect effects of ionizing gamma radiation on a cell. Absorption of ionizing radiation directly disrupts atomic structures and producesbiochemical changes in the cells. Indirectly, the radiation may generate ROS and RNS through radiolysis of cellular water that contribute to persistent alterations in lipids, proteins, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) (Copied from Azzam et al., 2012).

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In humans, the persistence of such stressful effects in progeny cells has profound implications for long-term health risks such as emergence of a second malignancy after radiotherapy treatments (Cucinotta and Chappell, 2010). Increasing evidence also supports the role of chronic oxidative stress in the progression of degenerative diseases and radiation-induced late tissue injury (Azzam et al., 2012 and Sharma et al., 2012).

Figure 21. Ionizing radiation (IR) induces targeted and non-targeted (bystander) effects. Communication of stress-inducing molecules from cells exposed to IR propagates stressful effects, including oxidative stress, to the bystander cells and their progeny. The induced effects may be similar in nature to those observed in progeny of irradiated cells.(Copied from Azzam et al., 2012).

Although there are many common mechanisms of response of organism and cells to irradiation and other stresses they encounter, the main difference is the extent of DNA damage (Ravanat et al., 2001). However, these differences are mostly attributed to high dose rates. In cases of low dose radiation, direct effects of irradiation such as clustered DNA damage and DNA double strand breaks are minimal while the indirect DNA damages caused by the induction of ROS and RNS becomes major problem (Ravanat et al., 2001). For high doses, adverse effects accumulate in the cells in a deterministic manner that depends directly on the amount of the dose. However, for low doses the effects are stochastic, non-linear on the amount of the dose, and depend mainly on the efficiency of the stress response’s protective mechanisms (Moskalev et al., 2011).

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4.1.2 Effects of gamma irradiation stress on Kinetoplastids Similar to other organisms, the oxidative stress induced by gamma ionizing irradiation has profound effects on kinetoplastids. Single and double-strand breaks and base damage may occur in the kDNA after subjected to a large amount of gamma radiation-induced oxidative stress (Takeda et al., 1986 and Regis-da-Silva et al., 2006). Since kDNA replication always takes place earlier than mitosis, cell reproduction and proliferation is hindered once the DNA is damaged, suggesting that a viable kDNA is needed for cell division (Achim et al., 2002). Nevertheless, the oxidative stress induced by the ionising gamma radiation is observed to alter kinetoplastid gene expression in the first 96 hours after gamma radiation, when DNA repair has already been completed (Grynberg et al., 2012). Among the genes that are highly expressed, categories of members of the retrotransposon hot spot gene family and kDNA are the most up-regulated genes (Grynberg et al., 2012). However, functional gene categories related to basal metabolism, translation and protein degradation processes tend to be repressed during this time. Other researchers have reported the increased expression of Rad51 mRNA in T. cruzi after irradiation and have associated it with the resistance to ionising radiation observed in these organisms (Regis-da-Silva et al., 2006).

Apart from the inhibition of proliferation and decrease in the infectivity, gamma irradiation was also been observed to cause fragmentation of chromosomes in L. major and consequently produce changes in the karyotypes of these organisms (Seo et al., 1993). The inhibition of proliferation and decrease in the infectivity might be because of destruction of the parasite’s chromosomes. Certain doses of gamma irradiation are able to destroy the infectivity of the parasites but not their viability. Mice and chicken-embryos repeatedly inoculated with the irradiated T. cruzi parasites could not get infected (Brener, 1962 and Chiari et al., 1968).

Resistance to the ionizing gamma radiation varies within the pathogenic kinetoplastids as between other organisms. Irradiation doses of higher than 1000 Gy have shown to inhibit the mobility and reproductively of culture forms of T. cruzi (Silva et al., 1967 and Chiari et al., 1968). Blood forms of T.brucei gambeinse are sensitive to gamma radiation doses higher than 120Gy (Halberstaedter, 1938) while L.major culture forms can endure gamma radiation doses up to 500 Gy (Seo et al., 1993). It is possible that the mechanisms behind these parasites radiation resistance maybe part of the responses against the stresses the organisms face such as changes in temperature, pH and osmolarity in the insect’s saliva and gut (Kollien and

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Schaub, 2000). Inaddition, blood digestion by the vector may be sources of heme molecules required for the Fenton’s chemical reaction that produce reactive chemical species and consequently causing oxidative stress (Kollien and Schaub, 2000).

Although C. fasciculata parasites have been used previously as a model organisms to study kDNA replication and repair mechanisms of the pathogenic kinetoplastids (Saxowsky et al., 2002, Shapiro and Englund, 1995), no any study has investigated the responses of these parasites to gamma irradiation and that it is still unclear to wether the parasites are Trypanosomes or Leishimania—like, in terms of their DNA replication and repair mechanisms after stress. We therefore initiated studies to investigate theresponses of C. fasciculata parasites to gamma radiation. We particularly investigated the effect of the irradiation on the cell growth, metabolic viability, motility and morphology as parameters of our investigation. In the long term, this will provide basis in understanding the post- irradiation DNA damage and repair mechanisms in C. fasciculata as a model for the corresponding systems in the pathogenic kinetoplastids.

4.2 Experimental procedures 4.2.1 Parasites strain and cell culture The C. fasciculata promastigotes clone HS6 was used in this experiment and maintained in a culture media as previously described in methods section of chapter 2. The parasites were sub-cultured every 2-3 days to ensure log growth phase for subsequent experiments.

4.2.2 Irradiation of parasites C.fasciculatalog phase choanoamastigotes in tissue culture plastic flasks were irradiated with ionising gamma rays from a Cobalt-60 irradiator at the University of St-Andrews, School of Medicine Radiation facility. This apparatus has a dose rate of 2.51 Grays per minute (Co-60 half-life is 11.833 years).

4.2.3 Growth experiments A total of 200ml medium was inoculated with log phase C. fasciculata choanoamastigotes to a final density of 2x106 cells/ml. The medium containing cells was then divided into five cell flasks each containing 40 ml. The four flasks were irradiated at room temperature with varying gamma dosages (250, 500, 750 and 1000 Gy); the non-irradiated control flask (0 Gy) was left at room temperature the same length of time as the irradiated flasks. Cells were

63 counted using a haemocytometer immediately after irradiation and at 24 hours interval for 5 days post-irradiation to generate the growth curve. 4.2.4 Parasites Viability experiments Two techniques – the Trypan blue exclusion and the Resazurin (Alamar blue) reduction methods were employed to determine the viability cells after exposure to the radiation. For the Trypan blue exclusion test, 100 µl of cells was suspended in an equal volume of 0.4% Trypan blue solution. A 20µl cell suspension was thenloaded into a haemocytometer and examined immediately under a microscope at low magnification. The blue stained cells were counted and compared against the total number of cells counted to determine the structural viability using the equation: % viable cells = [1 – (Number of blue cells counted ÷ Number of total cells counted)] × 100. For the Alamar blue reduction assay, aliquots of 25% (10 ml) of the medium containing the irradiated cells, control cells (100% viability) and the cell-free medium were inoculated in the 96 well plates (200 µl/well) and incubated at 27oC in a shaker incubator for 68 hours.After 68 hours, 20 µl of Alamar blue (0.125 mg/ml) was added to the wells and the plates further incubated for 4 hours. Fluorescence measurements were performed with the Spectra Max Gemini XPS Microplate reader (Gemini XPS, Molecular devices) at excitation wavelength of 530nm and 590 nm emission wavelength of 530 nm. The experiments were performed in duplicate and the results were averaged over eight replicate wells and normalised using an equation: Activity (%) = [1 − (FUntreated– FTreated) / (FUntreated– FCell free)] ×100, where Ftreated corresponds to the emitted fluorescent signal expressed in arbitrary fluorescence units for the treated; and FUntreated andFCell free correspond to the mean fluorescent signal of the untreated and the cell free wells, respectively. 4.2.5 Parasites motility estimates A conventional wet mount technique was used to estimate a fraction of the parasites that were motile after exposed to ionising gamma radiation stress. Briefly, a 20 µl of cell suspension was pipetted onto a clean microscope slide and a coverslip gently lowered onto the sample. The slide was immediately examined using a microscope with a 20X objective. At least ten widely spaced fields were examined to provide estimates of percentage motile cells. 4.2.6 Parasites morphology To assess the parasites morphology after irradiation, cells were stained with Giemsa solution (Sigma-Aldrich) using a standard Giemsa staining protocol and visualised on a microscope. Briefly, slides containing cells were fixed in methanol for 5 minute and stained with Giemsa solution for 30 minutes. The slides were then washed gently with deionised water and air-

64 dried prior to microscopy. At least 10 widely spaced fields were examined per sample and 5 images were taken for morphology evaluation. 4.3 Results and Discussion In order to ascertain the response of C. fasciculata choanoamastigotes to the ionising gamma radiation, the cells were exposed to varying gamma dosages with all finishing at the same time experimental set-up. The non-irradiated control flaskwas left at room temperature the same time duration as the irradiated flasks to generate the growth curve as shown in Fig. 22. A clear correlation was observed between gamma raadiation doses and the decrease in the cell growth. The control non-irradiated cells reached early stationary stage of growth within 48 hours, more quickly than the irradiated cells. Remarkably, we found that a gamma dose of 1000 Gy was able to arrest the growth of C. fasciculata the first 24 hour of post-irradiation (Fig. 22). Culture forms of T. cruzi were observed to endure gamma radiation doses as high as 1000 Gy while their blood stream forms could only tolerate doses not more than 300 Gy (Silva et al., 1967 and Chiari et al., 1968). In contrast, bloodstream forms of T.brucei gambiense and cultured forms of L.major can only endure gamma doses of up to 120 and 500 Gy (Halberstaedter, 1938 and Seo et al., 1993), respectively. Surprisingly, a gamma ray dose of 500 Gy was able to arrest the growth of T. cruzi for 96 hours (Vieiraet al., 2014), longer than what have been currently observed with C. fasciculata parasites, which are able to resume normal growth just after 24 hours post-irradiation with as much as 1000 Gy. The C. fasciculata postirradiation growth kinetics might therefore be similar to the observed post- irradiation growth kinetics of a radiation resistant bacterium D. Radiodurans, which is also able to resume normal growth within 9-24 hours due to its robust DNA repair machinery (Liu et al., 2003). This observation may suggest that C. fasciculata parasites posses active post- irradiation recovery machinery compared to T. cruzi parasites. One might agree that since C. fasciculata parasites grows almost everywhere mostly in the harsh environments and unprotected from the UV from the sun, the parasites have developed rapid recovery mechanisms to cope up with the oxidative stresses in their environments. A comparative and systematic genome-wide investigation of the genes and pathways involved in these unique parasites post-stress recovery mechanisms would therefore be useful for further understanding of how other kinetoplastids respond to and recover from such similar stress.

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Figure 22. Growth curve of C. fasciculata choanoamastigotes after exposed to varying ionising gamma doses. Cultures were microscopically monitored and counted using a haemocytometer at 0 hour post-irradiation and 24 hours intervals for 5 days. Average count was obtained from two measurements per sample.

To determine the viability of the parasites at 0 hours and at 72 hours of post-irradiation, two techniques; the Trypan blue dye exclusion and the Alamar blue (Resazurin) reduction methods were employed, respectively.

The Trypan blue dye exclusion test has been widely used to determine the number of viable cells present in a cell suspension (Strober, 2001). It is based on the assumption that live cells possess intact cell membranes that exclude certain dyes such as Trypan blue whereas dead cells do not. Viable cells therefore, have a clear cytoplasm whereas a non-viable cell has a blue cytoplasm (Strober, 2001). In our experiments, no significant differences in cell viability after exposure to different radiation doses were observed at 0 hours (p≥0.05) (Fig. 23a). This observation is consistent with Emmet. (1950) and Chiari et al. (1968), who reported that some gamma dosages can only destroy the parasites infectivity but not their viability. This may suggest that unlike the DNA, which is a well-known primary target of irradiation damage, cell membranes are perhaps not altered with the gamma radiation induced oxidative stress. However, the decrease in the cell permeability due to the irradiation itself should not be ignored as a possible cause of this observation. Perhaps the irradiation itself rendered cells less permeable to the dye as demonstrated by Khale and his colleagues (Khare et al., 1982),

66 where the uptake of several amino acids was reduced in Candida albicans following exposure to various gamma dosages. The cell viability or rather the cell membrane integrity of post-irradiation when assessed with Trypan blue or any other dye exclusion method should therefore be interpreted with caution unless assessed in parallel with other specific parameters of membrane damage such as sulfhydryl content, potassium ion permeability, sodium ion and many more.

Alamar blue reagent has been widely used over the past 50 years in studies on cell viability and cytotoxicity in a range of biological and environmental systems (Rampersad, 2012). Live cells maintain a reducing environment within the cytosol of the cell. Resazurin, the active ingredient of alamar blue reagent, is a non-toxic, cell permeable compound that is blue in colour and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in colour and highly fluorescent. Live cells are able to convert resazurin to resorufin in proportional to the cell density, and thus increasing the overall fluorescence and colour of the media surrounding cells while dead cells. In our experiments, a clear correlation was observed between gamma dosages and the decrease in the cell viability after 72 hours of post-irradiation when assessed with alamar blue reduction assay (Fig. 23b). The dose dependent decrease in the viability of C. fasciculata parasites currently observed is consistent with other studies on the pathogenic kinetoplastids (Chiari et al., 1968and Regis- da-Silva et al., 2006).

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Figure 23. Viability of C. fasciculata choanoamastigotes post-irradiation. (a) Viability at 0 hours assessed with Trypan blue exclusion test. (b) Viability after 72 hours of irradiation assessed with Alamar blue reduction assay. Values represent the mean±SE of two experiments each performed with eight replicate wells.

The motility of the parasites was not significally affected with gamma radiation doses (up to 1000 Gys) (Fig. 24). Intriguingly, the parasites were very motile irrespective of their gamma irradiation doses when observed under the microscope immediately after irradiation. However, similar studies on T. cruzi have reported that the mobility of the parasites were rapidly affected with gamma dose of 4660 Gys; employing doses of 3500, 2450 and 1550Gys, the mobility could be observed up to 72 hours post-irradiation, after which it gradually decreased (Chiari et al., 1968). We can therefore speculate that doses less than 1550 Gys could perhaps have little or no effect on the motility of either T. cruzi or C.fasciculata.

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Figure 24. Motility of C. fasciculata choanoamastigotes at 0 hours of post-irradiation. A wet mount was prepared for each sample immediately after irradiation to determine cells motility. Motility was calculated as percentages of mobile cells out of all cells counted.Values represent the mean±SE of three counts.

We also did not observed significant morphological changes of the C. fasciculata parasites after exposure to gamma doses up to 1000Gy, contraly to T. cruzi parasites that are able to maintain their morphologies when exposed to gamma doses not exceeding 600 Gy. However, at 0 hours post-irradition, cells subjected to 250 Gy and 1000Gy were long and slender and stained less dense than the controls (Fig. 25). Structures such as the nucleus, kinetoplast and flagillum could be observed after 120 hrs of post-irraditon indicating a recovery. Future studies should aim to use more sensitive stains and powerful tools like electron microscopy to precisely assess morphological changes of individual ultrastructural features of the parasites after irradiation.

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Figure 25. Morphology of C. fasciculata at 0 hours and 120 hours of post-irradiation. Cells were stained with 10% Giemsa solution. At least 10 widely spaced fields were examined per sample and 5 images were taken for morphology evaluation.

In general, although it was also not possible to conduct a side-by-side comparative study of the susceptibility of C. fasciculata and the pathogenic kinetoplastids to gamma irradition, C. fasciculata appears to be more like T. cruzi in respect to the resistant to gamma radiation and suggests that these parasites may perharps posses an enhanced postirradiation recovery mechanism as compared to the pathogenic kinetoplastids. C. fasciculata may therefore be a suitable organism fundamental for studies aimed at understanding the rapid kDNA repair mechanisms and protein expression profiles in the kinetoplastid pathogens when exposed to harmful environments.

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5. Chapter 5: General discussion and principal conclusions

5.1 General Discussion This project aimed at exploring the utilization of C. fasciculata as a convenient model organism to study the biology of the pathogenic kinetoplastids and its suitability as a drug discovery vehicle.We specifically aimed to; (i) Develop and validate an expression system that can be used to purify protein complexes from C.fasciculata. (ii) Develop C. fasciculata as a non-pathogenic compound screening model for kinetoplastid diseases. (iii) Study the effects of ionizing Gamma radiation on the model kinetoplastid C.fasciculata.

One may argue on the significance of conducting these studies in C. fasciculata parasites, given that many tools are currently available to do such studies directly in trypanosomes or leishmania species. Nevertheless, C. fasciculata is a monogenic parasite of insects and therefore may not have some important biological pathways found in the digenic kinetoplastid pathogens. For example, the biological payways that help the pathogenic kinetoplastids to survive in stressful environments found in the veterbrate hosts. This may make C.fasculata limited in the number of pathways representing potentially druggable targets shared with the pathogenic kinetoplastids. In addition, conducting studies in C. fasciculata and then validating them in the actual pathogenic kinetoplastids seem to be a very long process than conducting these studies direct on the kinetoplastid pathogens. However, although this might be the case, studies on the pathogenic kinetoplastids have for been hampered by the need to culture these highly infectious organisms safely in the laboratory. The dedicated containment level 3 (cat 3) facilities are needed for this purpose but these are very expensive to build, maintain and equip as experimental apparatus cannot be moved in and out of the cat 3 lab without rigorous decontamination. The trypanosomes T. brucei brucei, the causative organism of liverstock trypanosomiasis and leishmania tarentolae, a protozoan parasite of Gecko has been widely used in studies as safer altenatives of their human pathogenic couterparts. However, unlike these models, C. fasciculata have a shorter generation time and can beeasily grown to high densities using comparably less expensive media in a standard laboratory, thus reducing the time required to harvest adequate numbers of parasites for some large-scale applications. Nevertheless, Crithidia is an excellent example of a parasite that can easily be isolated and cultured outside of its normal host organism. It is an ideal model to study the biology of not only a single kinetoplastid but rather three pathogenic kinetoplastid (T.brucei, T. cruzi and leishmania species). It is also important to

71 notice that the T. brucei brucei, which was traditionaly known to be human non-infectious organism, has been reported to cause disease in humans (Deborggraeve et al., 2008). Moreover, unlike the trypanomes models, C. fasciculata is easly amenable to molecular genetic and biochemical analysis and itscomplete genome has already been determined and is publically available online to facilitate these studies.

In this study, we have confirmed that C. fasciculata can be cultivated in high yields (to high cell densities) using our inexpensive serum-free media and can be handled in a standard laboratory without specific bio-safety precautions. By modifying an existing plasmid from Tetaud and his colleagues (Tetaud et al., 2002), we have successfully constructed plasmid pNUS-PTPcH, which can be utilised for expressing and purifying kinetoplastids protein complexes. Protein expressed from pNUS-PTPcH has the PTP tag, a variant of the TAP tag (but with CBP in TAP is replaced by the protein C peptide). The PTP tag overcomes some of the limitations of the conventional TAP method as described elsewhere (Schimanski et al. 2005; Schimanski et al., 2003; Drakes et al., 2005; Palfi et al., 2005). We have shown that C. fasciculata can be efficiently transfected with pNUS-PTPcH by electroporation with Amaxa program X-014 (Burcard et al., 2007), and that the encoded PTP tagged proteins can be readily detected by Western blotting. The pNUS-PTPcH plasmid was maintained as a circular extrachromosomal DNA and conferred a hygromycin resistance on C.fasciculate parasites. As a proof of concept, we have successfully cloned, expressed and isolated C. fasciculata proteins and their interacting partners though TAP. PTP purification from cell lysates expressing RFC3-PTPcH identifies its interacting proteins RFC1, RFC2, RFC4, RFC5 and RAD17, while PTP purification from cell lysates expressing RRP4-PTPcH identifies RRP6, EAP1, RRP45, RRP40, RRP41B, CSL4, EAP2, RRP41A and EAP4 that have been previously isolated in T.brucei exosome complex (Estevez et al., 2001).

Since RFC complex has not been characterised in any kinetoplastid organism, the identification of its associated subunits in C. fasciculate will provide a basis for future studies on these complexes in other kinetoplastid pathogens. In particular, the identification of Rad17-RFC as the only alternative complex in our immunoprecipitation experiments may suggest that this complex co-exist with the RFC complex in C. fasciculata contraly to what have been observed in other organisms such as S.cerevisiae (Green et al., 2000). Nevertheless, although Rad 17 and other large RLC subunits Ctf18 and Elg1 have similar sequence to

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RFC1 but are considered non-essential genes (reviewed in Kim and MacNeill, 2003), the identification of Rad17 as only RLC subunit in our experiments may also suggest that perharps Rad17 have some essential roles in these specific parasites and should be properly explored as a potential target for new drugs.

Despite the conserved features and components, the exosomes of different eukaryotes are generally not identical. The genes of the six Rnase PH in human exosome complex do not reveal any orthologous in yeast or trypanosomes and there are no homologs for Rrp43p or Mtr3 in T. brucei (Estevez et al., 2001). Moreover, the T. brucei and yeast exosome complex have the RRP44 that is not present in human exosomes (Estevez et al., 2001). We also did not find C. fasciculata homologs of T. brucei RRP44 in both our database searches and immunoprecipitation experiments and never has itbeen identified in Leishmania species (Cristodero et al., 2008). This may suggest that kinetoplastid organisms, RRP44 is perharps conserved in T. brucei. All components of the yeast and human exosome except Rrp6p are essential for viability. Contraly, depleting RRP6 in trypanosomes caused a loss of both RRP45 and RRP4, suggesting that unlike in yeast or human cells, RRP6 may have an important role in the trypanosomes (Estevez and Clayton, 2010). Depletion of the Rnase PH domain proteins RRP41 and EAP1 disassembled the T.brucei exosome complex but had no effect on the humun exosome complex (Estevez and Clayton, 2010). Functionally, the T. brucei RRP4 exosome subunit has a processive exonucleolytic activity while its yeast counterpart has a distributative mode of action (Mitchell et al., 1997). The observed uniqueness in the composition and functions of trypanosomes exosomes as compared to yeast and more importantly human’s exosomes makes them ideal drug targets. In particular, utilising the RRP44 as a target protein in human trypanosome infection implies that the drug will specifically inhibit the activity of RRP44 in the trypanosomes. Using the trypanosomes exosomes subunits that have homologues in the host (humans) as drug target will result into a drug cross attacking the host essential exosome subunits causing disastrous effects. More studies are therefore, needed to characterise the molecular mechanism of RRP44 subunit to be considered as a drug target and perhaps a potential biomarker to aid in diagnosis of trypanosomes.

Although we have shown that the pNUS-PTPcH plasmid can be utilized to express and isolate kinetoplastids proteins in C. fasciculata, a few points need to be critically looked at for its wider utility. For example, western blots of extracts from cell lines expressing the empty pNUS-PTPcH revealed a very weak protein band for a PTP tag. However, the fact that

73 a protein band though still weak was observed in the immunoblots of its extract after 4 weeks make us speculate that perhaps a few copy numbers of pNUS-PTPcH plasmid are maintained in the parasites. Future work should therefore consider quantifying copies of pNUS-PTPcH maintained in the parasites. Epitope tags and stringency conditions applied in the purification process might interfere with functions of the proteins as shown in different studies elsewhere. Whether the protein subunits we purified in this study are functional or not is still unknown. It is also imperative to point out that although we have demonstrated the successful PTP purification of replication factor C and exosome complexes proteins, we did not quantify the yield of the proteins purified neither did we manage to pull down other novel proteins as initially anticipated. Future work should therefore consider monitoring the purification efficiency by quantifying the yield of the proteins at each step of the purification process. In addition, it will be important to consider optimizing purification conditions such as buffers used for preparation of cell-free extracts for efficient extraction and purification. Since we used a dounce homogenisation to lyse the cells, other cell lysis methods such as sonication should be considered in the future for comparisons. Nonetheless, while a C-terminal tag has the inherent advantage that only fully synthesized proteins carry the tag, some proteins are inactivated by a C-terminal tag. These proteins may accept a tag fusion at the N-terminal and may improve the purification efficiency (Schimanski et al., 2005). Future studies should therefore consider altenative-tagging ways to compare the efficiency of each tagging on purification.

We also did not control our purification experiments to avoid the co-purification of contaminating proteins such as the major cytoskeletal proteins α and β tubulin that bind to the resins non-specifically. Although we used the C. fasciculata cell line expressing the empty pNUS-PTPcH as a specificity control, the ideal control experiment was to generate a cell lines which expressed the PTP fused to an unrelated protein, which is preferentially expressed in the same cellular compartment as the protein of interest and then prepare the extract and perform TAP purifications and protein analyses in parallel. In this case, we would have excluded proteins that potentially interact with the TAP tag itself. Since we have cell lines for pNUS-DCC1-PTP construct (DCC1 is a subunit of the alternative Ctf18-Dcc1-Ctf8 replication factor C complex) and pNUS-Tfb4-PTP construct (Tfb4 is a subunit of theTFIIH complex), we plan to make further optimisations in our purification process and conduct more TAP experiments with these cell lines with an anticipation to pull down some novel

74 proteins which will be further characterised and validated as potential drug targets in the actual pathogenic kinetoplastids.

One promising approach for identifying new compounds for the drug development pipeline for treatment of patients with diseases caused by kinetoplastids is by HTS of compound collections. Simple in vitro cell culture systems for axenic growth of pathogenic kinetoplastids has led to the exploration of a various cost effective whole cell assay formats to serve as tools for HTS with the aim of assessing a large set of chemical libraries and prioritize newly synthesised analogues in hit-to-lead and ultimately lead optimisation phases of the drug discovery process (Muskavitch et al., 2008). Considering that C. fasciculata shares a variety of biochemical mechanisms such as polyamine synthesis and methionin salvage with the medically and veterinary important pathogenic kinetoplastids, Tanasor and collegues initiated work of utilising a non-pathigenic kinetoplastid C. fasciculata to screen medicinal plants that could posses cross activities against the pathogenic kinetoplastids (Tanasor et al., 2006). Extending this work, we have currently developed a simple, robust and reproducible alamar blue reduction--C. fasciculata based phenotypic screening assay that can facilitate the process of predicting potential anti-kinetoplastid compounds, which can be later followed up against the pathogenic kinetoplastids. We have shown that the developed assay fulfils the necessary and desirable criteria for a HTS. Although this assay has been developed in a 96-well format, it may be amenable to automated liquid handler and used in the 384-well formats. Utilising the developed assay, we have identified attractive chemical scaffolds in the Open access chemical boxes that will be considered for follow up testing in the actual pathogenic kinetoplastids. In particular, we have identified a Pyrimidin-4-amine chemical scaffold which has derivatives with well known kinase and cytochrome P450 inhibitors (Gunatilleke et al., 2012 and Pena et al., 2016), the quinazoline-2,4-diamine scaffold which are also well referenced in the literature as folate synthase pathway inhibitors in Leishmania,Ttrypanosoma and Plasmodium (Pez et al.,2003; Khabnadideh et al.,2005; Muller et al.,2013) and the 5-nitrofuran-2-yl derivatives which are substrates for type I nitroreductases of various parasites that metabolise them into nitrile toxic products (Hall et al., 2011) and are also inhibitors of Mycobacterium tuberculosis H37RV (Doreswamy and Chanabasayya, 2013). Nevertheless, our screening revealed the potency of compounds harbouring Quinolin-8-ol and 2-pyridinyl moieties previously reported for their antifungal properties (Musiol et al., 2006) and CYP51 inhibitors (Meissner et al., 2013 and Kaiser et al.,

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2015), respectively. Weather these compounds are hitting same targets shared betweenC. fasciculata and the pathogenic kinetoplastids remains an open question. However, considering the evolutionary proximity and the number of conserved cellurar pathways between C. fasciculata and the pathogenic kinetoplastids, one may agree that these compounds are likely to target the same pathways and therefore compounds with anti- Crithidial activities should be more likely to work against the pathogenic kinetoplastids. To our knowledge, no any fluorochrome viability assay protocol for C. fasciculata parasites has been reported. Therefore, it is impossible to make direct comparisons and conclusions that this assay is ideal than any other alternative viability detection methods currently available unless this assay was developed and evaluated in parallel with a similar viability detection method. However, the fact that C. fasciculata can be used to predict potential compounds against not only one specific pathogenic kinetoplastids is of high importance. Since the 96-well method may not be suitable for undertaking HTS of larger libraries because of intensive labour requirement and expenses, it will be of value to upgrade the Alamar blue reduction-Crithidia assay into a 384-well format. Although we managed to identify such attractive chemical scaffolds, it attempts to conduct further comprehensive optimisations and analysis of the near neighbour compounds was not successful, mainly due to the cost of purchasing the related compounds and time factors. Future work should therefore consider further structural optimisation and investigation of the identified chemical scaffolds prior to testing them against T. brucei, T. cruzi, Leishmania and mammalian cell lines. However, additional assays such as serum shift, time to kill and reversibility of compound effect of the structurally optimised compounds should be considered to provide further criteria for advancing them through hit-to-lead phase of the project.

Responses to the ionizing gamma radiation-induced oxidative stress varies form one organism to another and within the kinetoplastids. Generally, the primary target of gamma radiation in kinetoplastids is the kDNA (Genois et al., 2014). Although the effect of gamma ionising radiation on the kDNA damage and associated repair mechanisms in pathogenic kinetoplastids have been extensively studied, little is known on howC. fasciculata responds to such radiations. More importantly, despite being used as model organism to study kDNA replication and repair mechanisms in the pathogenic kinetoplastids previously (Saxowsky et al., 2002; Shapiro and Englund, 1995, Ryan et al., 1988), it is still unclear to wether C.

76 fasciculataparasitesbehave more like trypanosomes or leishmania when it comes to theirresponses to irradiation.In this study, we have demonstrated that compared to culture forms of T. cruzi, which undergoes growth arrest for 96 hours after exposed to 500 Gy (Vieira et al., 2014), C. fasciculata is able to recover and resume normal growth within 24 hours after being subjected to gamma ray dose as high as 1000 Gy. It is important to notice that theC. fasciculata observed post-irradiation growth kinetics might be similar to the observed post-irradiation growth kinetics of a radiation resistant bacterium D. radiodurans, which has also a shorter (9-24 hours) post-irradiation cell growth arrestattributed to its robust DNA repair machinery (Liu et al., 2003). This observation may suggest that Crithidia posses very active post-irradiation recovery mechanisms as compared to other kinetoplastids. However, the effects of gamma irradiation on C. fasciculata currently reported should be interpreted with caution mainly because of the weakness of our experimental design. Our main aim was to investigate the response of C. fasciculata to ionizing gamma radiation as compared to the other kinetoplastids. Therefore, the ideal experimental design would have to conduct the experiments in parallel with the other kinetoplastids of interests to avoid bias. Future work should therefore aim to conduct a well-designed comparative study of the susceptibility of these organisms to gamma irradiation to provide undoubted data on the responses of these organisms to irradiation. Nevertheless, for detailed assessment of morphological changes, the extent of DNA damage and the repair kinetics at the level of a single cell, future studies should consider utilising more powerful tools like electron microscopy and assays like comet assay aspreviously described in Lolenzo et al. (2013).

5.2 Principal conclusions The overall aim of this study was to explore the utilization of C. fasciculata as a convenient model organism to study the cell biology of the pathogenic kinetoplastidsand its suitability as a drug discovery vehicle. This study has confirmed that C. fasciculata parasites can facilitate various important aspects aimed at studying the biology of different pathogenic kinetoplastids, an imperative step towards the discovery of their respective new drugs, new diagnostic approaches as well as preventive mechanisms.

The current study reports the construction of plasmid pNUS-PTPcH that can be utilised to express PTP tagged kinetoplastids proteins in C. fasciculata for subsequent purification. As a proof of concept, we have shown that C. fasciculata can be efficiently transfected with this

77 plasmid to facilitate the isolation of two protein complexes: replication factor C and the exosome. We have demonstrated that the expressed PTP tagged-replication factor C subunit 3 (PTP-RFC3) co-purifies with RFC1, RFC2, RFC4, RFC5 and RAD17, and that the PTP tagged exosome subunit RRP4 co-purifies with RRP6, EAP1, RRP45, RRP40, RRP41B, CSL4, EAP2, RRP41A and EAP4. RFC complex has never been characterised in any kinetoplastid organism. Therefore, the identification of its associated subunits in C. Fasciculate may provide basis for future studies on these complexes in other kinetoplastid pathogens. In particular, the identification of Rad17-RFC as the only alternative complex in our immunoprecipitation experiments may suggest that this complex co-exist with the RFC complex in C. fasciculata contraly to what have been previously observed in other organisms. As a continuation of this project, we are planning to conduct further TAP experiments on various PTP-tagged kinetoplastid proteins with an anticipation to pull down some novel proteins that will be further characterised and validated as potential drug targets in the actual pathogenic kinetoplastids.

This study also reports the development of a simple and robust resazurin-reduction cell viability-screening assay with C. fasciculata that can be used to predict compounds with potential activities against the pathogenic kinetoplastids. The developed assay fulfils the necessary and desirable criteria for a HTS and therefore could speed up the process of hit-to- lead and ultimately lead optimisation phases in the drug discovery cascade. Utilising the current developed assay, we repurposed the open access chemical boxes for anti-crithidial compounds that have revealed attractive chemical scaffolds that will be followed up against the actual pathogenic kinetoplastids. We are considering developing a C. fasciculata cell line resistant to some of the identified scaffolds and use the whole genome sequencing and recombineering techniques to identify specific drug targets in C. fasciculata. Plans are also on the way to upgrade the 96-well format Resazurin reduction-Crithidia based assay into a 384-well format compatible for large compound libraries.

Nevertheless, the current study has revealed that C. fasciculata parasites behave more like T. cruzi than T.brucei or leishmania in terms of their responses to irradiation. However, compared to cultured forms of T. cruzi that undergo growth arrest for 96 hours after exposure to 500 Gy of gamma radiation, C. fasciculata is able to recover and resume normal growth within 24 hours after being subjected to doses as high as 1000 Gy. These findings form basis in understanding the kDNA repair mechanisms in the kinetoplastid pathogens when exposed to such stressful conditions. Moreover, since work have already been initiated to determine

78 changes in gene expression during DNA repair and cell recovery mechanisms following ionizing irradiation in T. cruzi, it might be very interesting to extend this work to C. fasciculata to find out if there is any overlap in the genes up-regulated or down-regulated following exposure to gamma radiation.

In general, we have shown that C. fasciculata can be utilised as a covinient and ideal model organism to study the biology and speed up the drug discovery cascade of the pathogenic kinetoplastids. In particular, the constructed protein expression vector, the developed screening assay and the observed responses of C. fasciculata to gamma irradiation will form basis for future studies aimed to discover novel drugs, new diagnostic approaches and preventive mechanisms for kinetoplastid diseases.

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7. Appendices

7.1 Appendix 1. The C. fasciculata serum free defined growth media recipe.

Ingredient In 1 Litre distilled water Yeast Extract 5 g Tryptone 4 g Sucrose 15 g Triethanolamine 4.37 g Tween 80 4.72 mL Haemin (2.5mg in 1 ml of 50 mM NaOH) 4 mL

The pH of the media was adjusted to 8.0 with either NaOH or HCl before adding Haemin (final concentration of 10µg/ml). The media was filter sterilised using 0.22µm stericups vacuum filters (Merck Millipore) and stored in the cold room.

7.2 Appendix 2. Extraction of C. fasciculata genomic DNA.

Ingredients Stock concentration In 1000 µl Final concentratio Tris-HCl 1 M 10 µl 10 mM NaCl 5 M 20 µl 100 mM EDTA pH 8.0 0.5 M 50 µl 25 mM SDS 10% 50 µl 0.5% Proteinase K 20 mg/ml 5 µl 0.1 mg/ml Distilled water 865 µl

A 1ml of media containing C. fasciculata cells in log phase was centrifuged at 2000g for 5 minutes. The cell pellet was then suspended in lysis buffer (see table below) to the final volume of 1000 µl. Cells were lysed and incubated at 56oC for at least 3 hours and the gDNA was precipitated with 500 µl of absolute ethanol. The DNA was then spooled on a sterile pipette tip and washed 2 times with 500 µl of 70% ethanol. The ethanol was allowed to air dry off the spooled gDNA and the gDNA was dissolved into 100 µl elution buffer pH 8.5 (from PCR clean up Kit) before storage in a +4oC fridge.

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7.3 Appendix 3. The designed forward and reverse primers that were used to amplify the ORFs of target subunits.

Protein Primers Sequences Descriptions subunit RFC3 CfRFC3-5Nde GGTGGTGGTGCATATGGCA To PCR amplify the ACTTCGAAG RFC3 insert sequence. CfRFC3-3Not GGTGGTGGTGGCGGCCGCC To PCR amplify the AGCGCTGC RFC3 insert sequence. RRP4 CfRRP4-5Nde GGTGGTGGTGCATATGTCG To PCR amplify the TCAGGAGTC RRP4 insert sequence. CfRRP4-3Not GGTGGTGGTGGCGGCCGCC To PCR amplify the CTGGCGGC RRP4 insert sequence. pC-Seq-F GCAAGGCGATTAAGTTGGG To sequence inserts Vector TAAC within PTP vector pC-Seq-R TGTTGTCCACGGCTTCATCG To sequence inserts TG within PTP vector

The first 10 are random bases followed by a NdeI or NotI sequence (in yellow) and ORF sequence of a target subunit gene. The primers pC-Seq-F and pC-Seq-R were used to confirm the fidelity of cloning process. Oligonucleotides were ordered from Integrated DNA Technologies (IDT).

7.4 Appendix 4(a). A conventional PCR reaction recipe and cycling conditions using Q5 High-fidelity DNA polymerase enzyme.

Reaction recipe of PCR using Q5 High-fidelity DNA Polymerase Component 50 µl reaction Final concentration Template DNA 5 µl 500 ng 5xQ5 Reaction buffer 10 µl 1x 10 mM dNTPs 1 µl 200 uM Forward primer(100 µM) 0.5 µl 1 mM Reverse primer (100 µM) 0.5 µl 1 mM Q5 High fidelity DNA polymerase 0.5 µl 0.02 U/µl 5xQ5 High GC enhancer(optional) 10 µl 1x Nuclease free water 22.5 µl Final reaction volume 50 µl Thermocycling conditions for PCR Step Temperature (0C) Time Initial Denaturation 98 30 sec Denaturation 98 10 sec 30 Cycles Annealing 70 20 sec

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Extension 72 45 sec Final extension 72 2 min All reaction components were assembled on ice and the reaction mixture was quickly transferred to a thermocycler preheated to the denaturation temperature (98oC).

7.5 Appendix 4(b). PCR reaction recipe and cycling conditions for a standard My Taq Red MIX.

Component 20 µl Reaction Template 5 µl Primers (4 µM each) 5 µl My Taq Red Mix,2x 10 µl Final reaction volume 20 µl Step Temperature Time Cycles Initial denaturation 95oC 1 min 1 Denaturation 95oC 15 sec Annealing 65 oC 15 sec 30 Extension 72 oC 10 sec

All reaction components were assembled on ice and the reaction mixture was quickly transferred to a thermocycler preheated to the denaturation temperature (95oC). 7.6 Appendix 5. Preparative restriction/diagnostic digest and ligation experimental set up.

Component Reaction (20 µl) Analytical/Preparative digest DNA 2 µl (1-20 µg) Buffer x2 10 µl Restriction enzymes (NdeI and NotI) 2 µl (1 µl each)

Sterile water 6 µl Dephosphorisation Antarctic Phosphatase Rxn Buffer (10X) 4 µl Antarctic Phosphatase 5 µl Sterile water 11 µl Ligations (20 µl reaction) 10 X T4 DNA Ligase Buffer 2 µl Vector DNA(~4 kb) 50 ng Insert DNA (~1 kb) 37.5ng Nuclease-free water upto 20 µl T4 DNA Ligase 1 µl

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7.7 Appendix 6. Recipes for solution and buffers used for PTP purification experiments.

Lysis buffer 150 mM sucrose 10 ml 300 mM potassium chloride 40 mM potassium L-glutamate 3 mM MgCl2 20 mM HEPES-KOH (pH 7.7) 2 mM dithiothreitol 0.1% Tween 20 TST buffer 50 ml 50 mM Tris-HCL (pH7.7) 150 mM NaCl 0.05% TWEEN

0.5 M glacial acetic acid 50 ml 1.43 mL Acetic acid in 50 mL d H20 PA-150 buffer 50 ml 20 mM Tris-HCl (pH7.7) 150 mM KCl 3 mM MgCl 0.1% TWEEN 1 mM DTT TEV buffer 20 ml 150 mM KCl 20 mM Tris-HCl (pH7.7) 3 mM MgCl 0.5 mM EDTA pH8.0 0.1% TWEEN 1 mM DTT PC-150 buffer 150 ml 150 mM KCl 20 mM Tris-HCl (pH7.7) 3 mM MgCl 1 mM CaCl2 0.1% Tween EGTA/EDTA elution 2 ml 5 mM Tris-HCl (pH7.7) buffer 10 M EGTA 5 mM EDTA Fix 200 ml 50% methanol 7% acetic acid Wash 100 ml 10% methanol 7% acetic acid

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7.8 Appendix 7 (a). Protein sequences of RFC complex subunits identified by Mass spectroscopic analysis. The residues that were found to be identical with the protein residues on database are highlighted in red.

RFC1

1 MSTLSFSTGI EAMTPVNTQS SATAAPRLSE LWADKYKPRS IAEMCYPSYA 51 NKLKAWLENF TPVGSPGDDP NKHHGVLLSG SPGVGKTTTV YVVARELGRT 101 VIEYNASDFR SRKSLRENVL DLISNRAFAA QATSYTRAVL LMDEVDGCDI 151 GGVGEVVKML FITKIPILCT CNDRWHPKLQ TLVKYVEDMR FSHPPCNIVA 201 NYLCERVLAR EGITLSKPLL QDIIKKSGSD IRNMLNNLQL WCLNRRSLEQ 251 RQLAECAAQA TKDGDAGLFD SAEYFLLQGT SRGERHSIAE MQACYYNSDL 301 IDMFVQENYL HYNPEPVDGR DWMTAVAQAA SSISRADAAQ RIMYYEQNWS 351 VSRFHVLSSS IAPCVYTRGK YETFMTGQQK FFDLQRPVKF PQWLGHNSTA 401 GKNRRLLRCV AMQASHPTRG ISGNQEDVAA DYMPNGWERP LTQPLAEKEK 451 DGIAEVIALM DQYNLMRDDW DLVQTLPHFR HMETPRQQPP VSITTAVKAA 501 FTREFNKTHR FDSFAKTTLK RTDKADEDDG IDEEEGESQK EGAGAKAGTK 551 GRVIADGVTA VTITGSDAAK PKAKTSAARK PRAKKSAANA AAAADDSGET 601 KPARKRAASA STRKPAKPAG KASKAAAGGK ARKRARVESS SESEVEISSD 651 SSSDSSDSE

RFC2

1 MSLSSQPVTK KAKTEAAASP AAAATPWIEK YRPRTLDEVE AQDEAVSALR 51 ACLKEGANMP HFLFHGPPGT GKTTSILAVA HELFGPDYIR SRVRELNASD 101 DRGINVVREK IKVFAQGAVS SGGSSVTQSD GKVYPVPGFK LIILDEADAL 151 LPDAQGALRR MMEDFSDVTR FCILCNYVSR IIDPIASRCA KYRFKPLVKT 201 ALYNRIQFVA NAEGIELSDA SLQALDSVSG GDLRLAIMHL QSAHKASGSD 251 LTREDFVSVS GSVPADAMQT YVAALVSRRL EDVIAVSRQL VAQGYAAAQV 301 LVQLQRYLVS AECPLNSAQR GRMMLKLCQT ERRLADGGDD YLQLLDMGSS 351 VCAS

RFC3

1 MATSKQAEDA KAGGSHLPWV EKYRPDNLDS VVAHEDILST LRHLMNSGNM 51 PHLLLYGPPG TGKTTTIKAC AYYLYGKDRV RANVLEMNAS DDRGIDVVRQ 101 QIREFSSTSS IFSMMGPSSS SGGGGNGGSG PLASFKLVIL DEADQMSHDA 151 QAALRRVIEK YTKNVRFCIL CNHINKVIPA LQSRCTRFRF APVKKSAMMP

201 RLKYVAEQEK VKYTTEGLAA AFRLSHGDLR RCMNTMQSSA LSADEITEES 251 VYRVTGNPTP AEVTAIVSDM LSGDFATSWA KVEVAVTQKG ISIADLAREI 301 HPIMMAMDLP QDCKCFLLMK LSDMEYYAAG GAREAAGLGG LLGAFQLVKE 351 AVTQRKPIKA VAGDCSA

96

1 MLCLARDLLL QNTDAATGAD KAGGKDILKD AVLELNASDD RGLDVVREKI 51 KLFAQTKKTL PKKFFTTGEG AETMEQVVHL HKIVLLDEAD SMTPAAQQAL

101 RRTMELHSST TRFAFACNNS SKIIEPIQSR CAVVRFKKLS DADILRRLVF 151 VIQQEKVSYT DDGLEALLYL AEGDLRQAMN SLQATHTGYG LVNADNVFKV 201 CDQPHPVLVE NIITACVTKR NIEEAHKEMN RLLNRGYAPA DVIATFFKVV 251 QTNARLFRSE LQQLEVLKVV GETTMRIAEG VGTSLQLAAM LARMIAAVEN 301 NQS

RFC4

RFC5

1 MLWVDRYRPK TLKEVELYPE LNDVLGRLAK AQDLPHLLFY GPSGSGKKTR 51 AMAVLHEIYG PSVYSVRLEH KSVQVSDSKV VDIATLSSPH HIDINPSDAG 101 NYDRVIVMQM IREIAQTVPL HTTASSAKAV PYKVVVLNEV DKMSRSAQHA 151 LRRTMEKYMK TCRLVLICNS TSRLIPPLRS RCLGIRVAAH SKDNLALAVQ

201 HVCEGESRPM PSPAFLNSLA LRSDGNLRRG LLMLEASAMT KVDWSGNGAA 251 IPQADWKLFL DEISHDILAE QTPKKLHEVR LKFYDLLAQC ISGETILKTL 301 LDSLLLAVPP KHQAALIQLA ATYDHNMKLG TKPILHLEAF VAGVMKLIKQ 351 Q

Rad17

1 MLNEVYAPTT VADLAWSRQK IVALSTLVRS TRSGAQNPRI LLLYGPPGCG 51 KLESLKVLLR EAPPAAASTT SKSKTPAPAP QVIEPPTTVS VFHTCEASST 101 AYSQFLQHVL SLCSGQLVGS ALMLTPKDMH GGRDTPSAPS DVQHAHIIKL 151 YGEPATHVLH RATVAFLRQY EALRLQAIRE EEQQQHQRRY LAKVLASPAS 201 PSTTLMDHLR RNLIFFVHTT HDSHNDKVDL GSALPAAVLQ SAAVELFHCT 251 PVTEINLKKR LRHILDTEAR RRANRSAQQR RADVAEATDV DDLFGIAPAL 301 SGSSAAPRRV AARGGAGSSR GKKGKENAKH APVTALHIPD AADVLDSLAL 351 DAIAAGSQGD IRQALLQVQW AALVPPGSST AASLVETVAD SSDVVWARLQ 401 HRRALAQAFA SGSSKADESS LVLSTKSVAP LAEACAAPQQ QDSTVAEDDD 451 GVVLLISSSS SEFDAPLPLS AAEVTRRQHL PSRSHEAATR KRSRSSENDV 501 VDVDDVGTTS KAAPPSAQAR ATDMLSLLDS QMNGAGESRA AAAASRGAAK 551 KLLRAAPVRR DGLAAKNNTD ADDGAAVLPD HRTVLPTTRD EYLGLSHATG 601 RLLSQKYSVD AVLDILNVPP RKMLDYLTNN QVRYFSDAQL PQYLVCAAAA

97

651 SEVDALRTAE FDGGYGGSAA ALRERRQLAD RTTAGESVGN VARLLDVIAL 701 QTFHRRYLVE QTAVQAPPGF TPQEPPPFLR SAYPRVRDVG STTNTTGPYM

751 TQRGEAVLEL LAGVSEHEWM EQFLLRLDSA VSASGAITSF SSIGRRRMPP 801 AASVVSSDAI FSQPALGLTS PSIRLDEVDI LREGLPDLLY RCGCTESVVM 851 DHYALAPYIV LNLPASSQPS AAAVASQPSP AVTPAGISSA ASGDSADAGG

901 APLSVARPRR TVFKFAASTP PPPPPTQLHS QPAPLSLRET HAARLSARRP 951 CTSLQLKILQ RGRDSAAATL RGDHFVLVAT ENIAEEGSMS EKGGVEERPW 1001 MPEGDDIEDD

7.9 Appendix 7 (b). Protein sequences of exosome complex subunits identified by Mass spectroscopic analysis. The residues that were found to be identical with the protein residues on database are highlighted in red.

RRP 41

1 MSRQKEYVSP AGLRLDGRRP LEARRMDIAF STLSGCDGSC DITLGRSKVC 51 ASVFGPRESV HRQEAKHDEV IITCEVAVAA FAGEVRRNPQ RRGRLSEDIS 101 AAVVQVARSV VLLPQYPNSQ IHIYLEVLQQ DGNEKIACIN AACLALVDAN 151 VAMRDAVCCT NVGLLDEHVL VDLTNEELRS QCPVIAAAFT GHDTRNIIWL 201 ETTSRLLPEA AIRLLKAAGQ SAKELFEGTV RGALVEHATQ ILALQS

RRP41B

1 MSSALSGQSA VTLSSSSSSS HPTAAATAGA SYTRRDGRTA LEIRGKEMRL 51 SEMADFDGSS WYAQGQTAVL VTLHGPTLAK NEEYDTCLVR VRVQHAHGLT 101 PSAGGAERAV YEEMKLEMLT RTDALELESL LESTIDAVVM RDRFPRCVLV 151 VDVVVVQDDG SLAAVALNAV MCALLDAGVP CRTTMAAVCV AAVTRAEDAA 201 AGDASRAVGS SLELLLDPTT AEETLGAGNT AAATAAGGEK ARSTVDATMA 251 EKGDLSGAAA AKLSLLRPDA LQGHYRCVST GVFVFSNPAC GGGVLAQLVR 301 RRSGGDSGTG ANTVSVEVYG QMMTLAERAA VVLFDFFRQC NVAE

RRP6

1 MPPKSAEASL PATKAVVSAV FGAVKDYSKL SAQIPADDFE YHLAFAGFRK 51 HIRDDSVGLV EVMDACCQML PKRRRTNLVA EEDPHSGAVH LAETQRNAVM 101 EAIDSLLENV DSLLDEVKGR KLDAQDQLSV TFGSELAVSA HHDASRGGSS 151 ASNAAGVVRL AHVRRPQLSF ETPVDNSAAP FTPTYRDASG VQHTGVAGEH 201 PFHDAIRAFS VPEAQMMPKA EIPPVPLETC PLSFVDTPDA MQAMVAKLLS 251 ASEIAVDLEH HDFYSYQGFT CLMQISTREE DFIVDCLQLR ASMGALAPVF 301 LNPSILKVFH GAREDVRWLQ KDFALYLVNF FDTGVALQTL HMPYSLAFAV 351 DHFCQVKLNK KYQTADWRVR PLPADMVHYA RQDTHFLLYV YDRLKALLLN 401 SEGRASVGNL LVHVYNESKQ LALQVYAKPN VDPAETYKLA LGRSLGGLTA

98

451 VQEEVAREIF NWRESAARDV DDSPTAVLHL SSVLAIASKL PTTAKDLLRC 501 CSPATAVLRA NVAHLVELVK KAVASSSEDF ENGVSGSGAG RHGKEEGSRH 551 HNYLDGAAEG SLEWAVYRSR CPTGVHRPMT GTLPSLASVV KTVTPAAVSV 601 SEQAALLSHT MPSSWFSAMS ALSRVLASRQ QHHVELPGAD VRAARQAAAA 651 KSLAGTADAA AAAAVAAEEE TAKAEVESVS SASGEGEQKD EEATGDLPAE 701 ADVAEASSVI ALDKKAFSIK QEYGVGAKSR FKKGEKGGAA KKKK

EAP1

1 MSVSAASISL AEVRAVQDGV ANDVREDGRT LLQRRPVYIT PRSSPSAVAA 51 VVGGSDGGDA AGVAQQSYSG SYVEVRASGT VVLAAATPTV VDGCATAAPS 101 PVSAADNADG AKEAAAAAPH DAGRGQLHIT IDAVPHVLDA YAGTVGGRNT 151 HRYRRDYLAF LAATIRAVFG AAQVQVQEQQ GVAEAEVVPE EREGAEDEVG 201 AGTVSSSAVA PAGSGRGDGE TSLASGFPAA DLYIGEGFGF RVHVDVHVLQ 251 CAGGNLFTAI AYAVHAALRS LQLPAVTLHR APGDGAGVSV EVDRSQPYRR 301 PVQWSQLPLL CVLLVSPTGH YVVDPTLREE WALPQQVHVA AGASGQVFYF 351 RYQQLPSRRG NRYQLQEARK ADAEACAAYV APPMALNLLD CWAVLSDAVY 401 VCQAMIHDCE VALQG

RRP45

1 MLLRSAAPVP ADALVQRNVE FARTAWRAGL RPDQREAHQL RMIEIEFPLL 51 ARDTVQVKCG NTIATASVTC DLVEPMPFRP KHGFFEVHAR QLLHERDPLD 101 QPKAVKQLSM YLTRLLSGSV VETEGLCVIP GRRVWSIAAE VLILNNDGNL 151 HDVAQWAVMA ALQHVRRPEL TIRGDDVVVH PPHERDPVPL SLHHIPLSFT 201 FAVCANPQQV QLAARAAALR RASPVSAGAA GQGSSDNAGE KEDGADASAW 251 SDDALQIVAD PSLEEAAAAA CTVSVAVNAE GHVCSLEKAD GCDVSLEHLE 301 QCMQVALQLT PPLLTQMQEA MAAHDVKRKA AVRSQFLWAQ KRLGIQAAGG 351 AGASQTQEEQ AAKKSKTE

RRP40

1 MSTHSPTLKS VSELVPLKGH VCLPGEPVLM VQSSAVVAVG GGLRLLAQPS 51 TATDASQDVA DVFLAEYCAP LQRSSHHLHT HVPRYTVATP ASRRYTPRHA 101 DPVIAVIARK VSQHYYYCYI GGSSLAYLEA IAFDGATKVS RPRLAEGDVV 151 YCYVKPRAAA SYVDGAAASS AAATAAAVSS GGEVELACTA AEVGLPPKDW 201 TSGEAVFGPL LGGRLLTLPL AYVRRLLAPL PATLSGEGPA VKRARVEGGG 251 GEAEEVPASY LLHLLGQRVP FEVAVGMNGL VWVKGLTSEA DATAAARRTV 301 AVSACISEAQ YDATRAEMEA RVESYFPS

99

RRP4

1 MSSGVVIVGD SICGGERIQK LNTSNDEVYL RGFNTFAGNN PSDIALVHEG

51 AGEIVAAING HIEVTDRVVS VKGLLPRYQP EIGDVVVGRI LEVTGNKWQV

101 DVNSTQTAIM LLSNVTEPGG MLRRRGRGDE LGMRQLFDQE DLVAAEVQRI

151 SPDGVVSLHT RAAEKYGRIG GFGVLVSVRP SLVKRAKHQF VELAEHHVRL

201 TIGMNGNIWV SRKEETADGT EDKEREAEAR QNVARVANCV KALGVAHIQI

251 HPATIEAAVA ASVEAGFSAF HVSLEKNRDA LLVSVHDAIG VKRRRQ

CSL4

1 MPVLVHTGAR VAPGDALFSS AAHVPTGTDA SAATAGDTVS DSDVIPGEGC

51 VVHYVEVPSE STGDSSRVRR HIVATRQGVA QWDGRLVSVF AAGATGTTAQ

101 LQGASTAVRS AVTGPRPGDT VHVRITRLSR LFAFGEITAV NWQWCSHRSA

151 AGASVSGVFK GVLRLEDIRP FRPTRDQLQP PPPTMAFALG DVVLAEVISQ

201 SDAHQYQLST VGEGFGVVES YVSTAEEHYS GRERVKLQHL PGRRDAMLVP

251 ATGAVVPRWC PLLP

EAP2

1 MSLPPNTGSI ELTAFRAHTS QLLARGERLD KRDFTTCRVP TVVREERAAE 51 APSSSSSGVV QTGINMANSG NLAAVMYTDS YGACMQCTVQ GLLGPPRPDR 101 PAAGRLNIHV EAPFVEQLGG GAATNYKSFQ YIISNGNADL PLRQLEGYIG 151 SVVDGCFDPT QLSIYDGEAC WVLNVTVTLL SFDGGLRAAS LHAVLAALHQ

201 LRLPRTRLPN GDVIESRRVR LSCLPTACTF GFLAGAQVRL LADTTAIEEY 251 VADGLLTIAV SESGEVVGVH QVGRCPLLAQ ALTAAVQQWT EQSASVRKAL 301 YG

EAP4

1 MTRLDGRQST EAVRAIHVAT NVLANCHSSA CVEIGQTRVL CGVRPPQQLV

51 QEYRGTRGRI SCQLHRSSAS SAAATVADNS ADRDMALALE GVAEQAVVLE

101 RIPQLLVEVL IEVLHDDGAV WDAAATALSA ALTAGGVEVY DTFTACSAAV

151 RPDGAIVVDL TQEEEAAATA RVVVCGGVSL GGVYYMCHLG ACEAATMAQL

201 VQAATKGMQV RKALLLEQIR NQ

100

7.10 Appendix 8. The full sequence of the constructed pNUS-PTPcH plasmid

AAGCTTACCGACAAGACCAGAATAGCGCTTATGTGTGTGTGTTTGTGTGTGGGTGTGTGTGCCCATGTGTGACAAAGAG GCCAGTGCTTTCTCTAAAGCAAGAGCGCGAGGTTGCTGGCTAAGGACTCTAAGTCCCTCACAGTGATGGCGTGTTGCAT TTTCTAAACCGTCAATCCAACACGTCGGTAGCATATCGGTTTTGTAGGCTCTTCTGGATACCTTCCCCAGTAAGACCGC CACTGCTTCGTGACTGAAGCACTGTAAATATCACTGAAACCGTCGCGTTGCTTTGTCTAATTATCACCGCCTTTGCCAG CTATCGCCTCAGGCTGCGCTCTTTGACTAATCCACTCACCACTTCTCTGCCTTCTTCCTCCGGTTGTTGTTGTGTCCTG CGTGTACATGGCGCGTGTCCTTTTCGAGCAAACAGCTGTCTTTTCTTTCACGATAACACACTCATATTAAACGCGAGTA TTACTCATCAGTCAACGTCACATTCCGCTCTGTCCACTTCGACCTTACACCTCTACTTGTCCAACTATCTTCCACTTGT CAAGCGAATTCCATATGCTCGAGGATATCGGCGGCCGCGAAGATCAGGTCGATCCTCGTCTTATTGATGGGAAATATGA TATTCCAACTACTGCTAGCGAGAATTTGTATTTTCAGGGTGAGCTCAAAACCGCGGCTCTTGCGCAACACGATGAAGCC GTGGACAACAAATTCAACAAAGAACAACAAAACGCGTTCTATGAGATCTTACATTTACCTAACTTAAACGAAGAACAAC GAAACGCCTTCATCCAAAGTTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGA TGCTCAGGCGCCGAAAGTAGACAACAAATTCAACAAAGAACAACAAAACGCGTTCTATGAGATCTTACATTTACCTAAC TTAAACGAAGAACAACGAAACGCCTTCATCCAAAGTTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAG CTAAAAAGCTAAATGATGCTCAGGCGCCGAAAGTAGACGCGAACTCCGCGGGGAAGTCAACCTGATAATAGGAATTCTG TATTACGCCGTTTTAAGAGCTCCTTCGACGCCTTTTTGTCCTTCAGCTGCTCCACGGTGACTCGCTTCTCTCTCTTTCC ACAGTGTCTCTTTTTTCTTTTCACTCTCTATACAAATGTGAGCGACCTCCTTTTCTGTACAACGGCCTTCCGGCGTGTG CTTTTCTCATCGACCTCTCCTCGCTTCTTGGGCACTCCTTCATCGAGCAAACAAGACGAGGGAGGTGACGCGATTGGTG AGACCACTACGAGTATGGACGGAGCTGTCTGATACCCCCTGTTTTTTATCTTGTACCCCGCTCTGCAGTGACGGTAGCC CGCTGCTGTGCTTCGGTATCGCCGCTTCATACGCTTCTCTCTTTTTTCAACGTGCGGCGCTGATTCAAAGGTTACCTCA ACGCGACCCGCCGCTGCATCCTTTGTTGCTCCTCTTGTCGCAAACAAACAAAAAACGTGCTGTGCTTTTTCCTTTATCG TGTCTTTTTGCAAAGTCTAGAGTTTACCGACAAGACCAGAATAGCGCTTATGTGTGTGTGTTTGTGTGTGGGTGTGTGT GCCCATGTGTGACAAAGAGGCCAGTGCTTTCTCTAAAGCAAGAGCGCGAGGTTGCTGGCTAAGGACTCTAAGTCCCTCA CAGTGATGGCGTGTTGCATTTTCTAAACCGTCAATCCAACACGTCGGTAGCATATCGGTTTTGTAGGCTCTTCTGGATA CCTTCCCCAGTAAGACCGCCACTGCTTCGTGACTGAAGCACTGTAAATATCACTGAAACCGTCGCGTTGCTTTGTCTAA TTATCACCGCCTTTGCCAGCTATCGCCTCAGGCTGCGCTCTTTGACTAATCCACTCACCACTTCTCTGCCTTCTTCCTC CGGTTGTTGTTGTGTCCTGCGTGTACATGGCGCGTGTCCTTTTCGAGCAAACAGCTGTCTTTTCTTTCACGATAACACA CTCATATTAAACGCGAGTATTACTCATCAGTCAACGTCACATTCCGCTCTGTCCACTTCGACCTTACACCTCTACTTGT CCAACTATCTTCCACTTGTCAAGCGTCGACATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCG GAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGG GCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCG GCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCAC AGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTTCAGCCGGTCGCGGAGGCCATGGATGCGAT CGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGT GATTTCATTTGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCG CGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTC CAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATAC GAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATC CGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGA CGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGT ACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCC TCAGCACTCGTCCGAGGGCAAAGGAATAGGGATCCAGCAGGCGGAGAAAGAGGGAAGTATAAGGCGGACGCATAGGTCG GAGTATCGAGAAAAAGAGGCAGAGATGGGTGGTGGCGGAGCGCCCCTCTCTGCCCTTCTCTGTTTTACTGTTCCCGCCA CGTCGCCAACTCCTCTTTTTGTTTGCTCTAATGTCGGTGCTATCCTCTTCCTCTTTTTACTCTCCGTTTTTCTTCCTTC CGTTTTGTCTTGTTATCACCGTTATTTTTCTTCTTCTTTCTTAGCCGATTTGGGTCTCCTGCCTACGGCAGCGGTGATG AGTCGCACTTCCGTCCCCCTTTCCTCTCCGTAAGTACTCCCTCGATGCCTCAGGCGCTTCTATTTTGCGGCACTGTGCT GACCACCTCCCACGTGTGCAGTGAGAGCGCCAGAGACATTCAGGAGAAGAGGGAAGAGGGGAAGTAAATACCAAAGCGA GGAAGATGTCTTTCTCGCTGCTTCTCATCCTGTTGACCGGTGTGCACGGCGGTGTGTCCTCTCGGCTTCCCTCCTCCTT CCCTCGCTCCCCTTTTTCTGTGTTTTTTCTTTCTAACATGATTGCGCCTGCTCTTTTTGCCCCGCAGCGTCGCAGTGGG TGTCACCTGCGACCGCCGTACGTTTTACTATATACATATTTGTTTCTGTGCTGATGCCATTTGTCCGCTACTCCATATC GTGCGTGTTTGTTTTCCAGTGGTGACGCCTTCCGCATCATTTTAATCAATTCGAGGCGAAAAGGGTGATTTGAGTGTTG GAGTGGGCTTTTTTATTTATTTGACTCCAAACATCTTTTCTTTTCCCGGTATCTTCGGGTCCGTCACGCAGGTCGGTGG GTGTGTCCGAGTGCGCTTCTTTACTCACGTGCCGCTGCGCACATACACAGATTTTAAAAGCACGCATACACGATCTGTG CCTTCAAAACTAATCAACAAAAACAAAAATACAAAAAAGACAAGAATAAGAGGTCAGCAACGCACCCACGGCTCCTTTC TTCCTGATCCACGTCGTGGCCGCTGTACGCTCTCAAACACGCTTTGGCGCTGATGCGTGCCTTTATACAAAGAACAAAG

101

AAGAAGCGAAAAACGGGAGCGTGTTGCTTTGGGGATGTATGCGTGCCACAGTTCTGCGGTACAATTCGTAATCATGGTC ATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAA TCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGT TTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCA TAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGC TCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCAC TCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCC CCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCC TTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGT AAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGC GGCATCAGAGCAGATTGTACTGAGAGTGCACGATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGC ATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAC GGCCAGTGCC

7.11 Appendix 9. Profiles and percentage anticrithidial activities of compounds in the MMV pathogen box

Plate barcode Position Compound ID Inhibition (%) at 100µM concentration PathogenBox_PlateA A02 MMV010764 92 PathogenBox_PlateA A03 MMV688472 67 PathogenBox_PlateA A04 MMV688416 59 PathogenBox_PlateA A05 MMV689758 87 PathogenBox_PlateA A06 MMV688796 79 PathogenBox_PlateA A07 MMV676526 19 PathogenBox_PlateA A08 MMV688553 28 PathogenBox_PlateA A09 MMV676501 73 PathogenBox_PlateA A10 MMV676449 87 PathogenBox_PlateA A11 MMV676412 94 PathogenBox_PlateA B02 MMV1110498 90 PathogenBox_PlateA B03 MMV000907 95

102

PathogenBox_PlateA B04 MMV688889 20 PathogenBox_PlateA B05 MMV688776 75 PathogenBox_PlateA B06 MMV688934 68 PathogenBox_PlateA B07 MMV676389 82 PathogenBox_PlateA B08 MMV676603 70 PathogenBox_PlateA B09 MMV676401 86 PathogenBox_PlateA B10 MMV102872 94 PathogenBox_PlateA B11 MMV676477 72 PathogenBox_PlateA C02 MMV084603 91 PathogenBox_PlateA C03 MMV688548 95 PathogenBox_PlateA C04 MMV688888 90 PathogenBox_PlateA C05 MMV690028 73 PathogenBox_PlateA C06 MMV688943 95 PathogenBox_PlateA C07 MMV053220 73 PathogenBox_PlateA C08 MMV676584 88 PathogenBox_PlateA C09 MMV676439 84 PathogenBox_PlateA C10 MMV676395 70 PathogenBox_PlateA C11 MMV676379 82 PathogenBox_PlateA D02 MMV687762 88 PathogenBox_PlateA D03 MMV1028806 94 PathogenBox_PlateA D04 MMV661713 17 PathogenBox_PlateA D05 MMV688793 90 PathogenBox_PlateA D06 MMV688942 95 PathogenBox_PlateA D07 MMV688554 72 PathogenBox_PlateA D08 MMV676555 85 PathogenBox_PlateA D09 MMV676383 86 PathogenBox_PlateA D10 MMV676444 77 PathogenBox_PlateA D11 MMV676409 95 PathogenBox_PlateA E02 MMV688514 87 PathogenBox_PlateA E03 MMV676350 72 PathogenBox_PlateA E04 MMV553002 94 PathogenBox_PlateA E05 MMV688797 88 PathogenBox_PlateA E06 MMV688756 70 PathogenBox_PlateA E07 MMV090930 88 PathogenBox_PlateA E08 MMV676431 42 PathogenBox_PlateA E09 MMV676571 76 PathogenBox_PlateA E10 MMV676445 94 PathogenBox_PlateA E11 MMV676589 80 PathogenBox_PlateA F02 MMV026020 91 PathogenBox_PlateA F03 MMV688471 88 PathogenBox_PlateA F04 MMV676388 95 PathogenBox_PlateA F05 MMV202553 76 PathogenBox_PlateA F06 MMV688936 92 PathogenBox_PlateA F07 MMV676476 64

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PathogenBox_PlateA F08 MMV676377 95 PathogenBox_PlateA F09 MMV676406 75 PathogenBox_PlateA F10 MMV676461 84 PathogenBox_PlateA F11 MMV676509 76 PathogenBox_PlateA G02 MMV688470 93 PathogenBox_PlateA G03 MMV688704 90 PathogenBox_PlateA G04 MMV188296 60 PathogenBox_PlateA G05 MMV688958 89 PathogenBox_PlateA G06 MMV063404 87 PathogenBox_PlateA G07 MMV676558 73 PathogenBox_PlateA G08 MMV688555 64 PathogenBox_PlateA G09 MMV676597 95 PathogenBox_PlateA G10 MMV676588 96 PathogenBox_PlateA G11 MMV676554 70 PathogenBox_PlateA H02 MMV688350 94 PathogenBox_PlateA H03 MMV688360 95 PathogenBox_PlateA H04 MMV099637 95 PathogenBox_PlateA H05 MMV688798 93 PathogenBox_PlateA H06 MMV676539 95 PathogenBox_PlateA H07 MMV202458 95 PathogenBox_PlateA H08 MMV676474 92 PathogenBox_PlateA H09 MMV461553 94 PathogenBox_PlateA H10 MMV676520 95 PathogenBox_PlateA H11 MMV676512 95 PathogenBox_PlateB A02 MMV676480 52 PathogenBox_PlateB A03 MMV652003 64 PathogenBox_PlateB A04 MMV000062 72 PathogenBox_PlateB A05 MMV006372 40 PathogenBox_PlateB A06 MMV688854 71 PathogenBox_PlateB A07 MMV011903 70 PathogenBox_PlateB A08 MMV020591 46 PathogenBox_PlateB A09 MMV020623 71 PathogenBox_PlateB A10 MMV020512 70 PathogenBox_PlateB A11 MMV688761 75 PathogenBox_PlateB B02 MMV012074 66 PathogenBox_PlateB B03 MMV676604 52 PathogenBox_PlateB B04 MMV002529 68 PathogenBox_PlateB B05 MMV687776 80 PathogenBox_PlateB B06 MMV687800 40 PathogenBox_PlateB B07 MMV020982 60 PathogenBox_PlateB B08 MMV020120 56 PathogenBox_PlateB B09 MMV676605 66 PathogenBox_PlateB B10 MMV007638 56 PathogenBox_PlateB B11 MMV021057 74

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PathogenBox_PlateB C02 MMV690027 68 PathogenBox_PlateB C03 MMV676600 63 PathogenBox_PlateB C04 MMV676382 62 PathogenBox_PlateB C05 MMV001625 70 PathogenBox_PlateB C06 MMV001493 41 PathogenBox_PlateB C07 MMV020136 17 PathogenBox_PlateB C08 MMV020710 40 PathogenBox_PlateB C09 MMV020517 52 PathogenBox_PlateB C10 MMV019721 63 PathogenBox_PlateB C11 MMV688763 80 PathogenBox_PlateB D02 MMV020537 50 PathogenBox_PlateB D03 MMV637953 69 PathogenBox_PlateB D04 MMV676536 67 PathogenBox_PlateB D05 MMV000063 54 PathogenBox_PlateB D06 MMV689255 56 PathogenBox_PlateB D07 MMV019838 68 PathogenBox_PlateB D08 MMV020520 51 PathogenBox_PlateB D09 MMV019234 23 PathogenBox_PlateB D10 MMV016136 28 PathogenBox_PlateB D11 MMV688762 29 PathogenBox_PlateB E02 MMV676386 64 PathogenBox_PlateB E03 MMV688773 64 PathogenBox_PlateB E04 MMV000011 59 PathogenBox_PlateB E05 MMV687775 76 PathogenBox_PlateB E06 MMV002817 76 PathogenBox_PlateB E07 MMV676442 40 PathogenBox_PlateB E08 MMV020152 50 PathogenBox_PlateB E09 MMV024397 20 PathogenBox_PlateB E10 MMV019807 65 PathogenBox_PlateB E11 MMV560185 25 PathogenBox_PlateB F02 MMV019189 46 PathogenBox_PlateB F03 MMV688774 78 PathogenBox_PlateB F04 MMV003270 14 PathogenBox_PlateB F05 MMV637229 76 PathogenBox_PlateB F06 MMV688853 82 PathogenBox_PlateB F07 MMV020321 62 PathogenBox_PlateB F08 MMV019087 8 PathogenBox_PlateB F09 MMV676528 62 PathogenBox_PlateB F10 MMV020320 34 PathogenBox_PlateB F11 MMV085210 73 PathogenBox_PlateB G02 MMV069458 68 PathogenBox_PlateB G03 MMV688991 51 PathogenBox_PlateB G04 MMV687801 58 PathogenBox_PlateB G05 MMV689480 78

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PathogenBox_PlateB G06 MMV003152 78 PathogenBox_PlateB G07 MMV006239 70 PathogenBox_PlateB G08 MMV000858 21 PathogenBox_PlateB G09 MMV006741 20 PathogenBox_PlateB G10 MMV688768 81 PathogenBox_PlateB G11 MMV000023 70 PathogenBox_PlateB H02 MMV676602 78 PathogenBox_PlateB H03 MMV000014 82 PathogenBox_PlateB H04 MMV687803 14 PathogenBox_PlateB H05 MMV668727 75 PathogenBox_PlateB H06 MMV019742 27 PathogenBox_PlateB H07 MMV009054 73 PathogenBox_PlateB H08 MMV006901 8 PathogenBox_PlateB H09 MMV020391 74 PathogenBox_PlateB H10 MMV676380 76 PathogenBox_PlateB H11 MMV688994 84 PathogenBox_PlateC A02 MMV675997 95 PathogenBox_PlateC A03 MMV676204 95 PathogenBox_PlateC A04 MMV687239 23 PathogenBox_PlateC A05 MMV688122 96 PathogenBox_PlateC A06 MMV688852 95 PathogenBox_PlateC A07 MMV687145 95 PathogenBox_PlateC A08 MMV688327 94 PathogenBox_PlateC A09 MMV008439 96 PathogenBox_PlateC A10 MMV595321 96 PathogenBox_PlateC A11 MMV687747 96 PathogenBox_PlateC B02 MMV020388 89 PathogenBox_PlateC B03 MMV688547 96 PathogenBox_PlateC B04 MMV688466 89 PathogenBox_PlateC B05 MMV687749 94 PathogenBox_PlateC B06 MMV688846 85 PathogenBox_PlateC B07 MMV054312 97 PathogenBox_PlateC B08 MMV689060 74 PathogenBox_PlateC B09 MMV689061 23 PathogenBox_PlateC B10 MMV689028 84 PathogenBox_PlateC B11 MMV688371 95 PathogenBox_PlateC C02 MMV688508 13 PathogenBox_PlateC C03 MMV688283 71 PathogenBox_PlateC C04 MMV687243 91 PathogenBox_PlateC C05 MMV687730 96 PathogenBox_PlateC C06 MMV687251 96 PathogenBox_PlateC C07 MMV687254 96 PathogenBox_PlateC C08 MMV688509 47 PathogenBox_PlateC C09 MMV688361 93

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PathogenBox_PlateC C10 MMV689029 62 PathogenBox_PlateC C11 MMV022236 95 PathogenBox_PlateC D02 MMV688410 68 PathogenBox_PlateC D03 MMV676048 96 PathogenBox_PlateC D04 MMV687703 96 PathogenBox_PlateC D05 MMV687248 87 PathogenBox_PlateC D06 MMV688125 89 PathogenBox_PlateC D07 MMV687188 95 PathogenBox_PlateC D08 MMV690103 95 PathogenBox_PlateC D09 MMV688124 16 PathogenBox_PlateC D10 MMV688845 68 PathogenBox_PlateC D11 MMV1030799 96 PathogenBox_PlateC E02 MMV675994 23 PathogenBox_PlateC E03 MMV676057 37 PathogenBox_PlateC E04 MMV687699 95 PathogenBox_PlateC E05 MMV687146 96 PathogenBox_PlateC E06 MMV687696 50 PathogenBox_PlateC E07 MMV687170 92 PathogenBox_PlateC E08 MMV690102 96 PathogenBox_PlateC E09 MMV689709 96 PathogenBox_PlateC E10 MMV021375 91 PathogenBox_PlateC E11 MMV1029203 90 PathogenBox_PlateC F02 MMV676053 60 PathogenBox_PlateC F03 MMV688179 96 PathogenBox_PlateC F04 MMV023969 96 PathogenBox_PlateC F05 MMV687138 52 PathogenBox_PlateC F06 MMV688262 96 PathogenBox_PlateC F07 MMV687189 89 PathogenBox_PlateC F08 MMV687807 48 PathogenBox_PlateC F09 MMV676478 87 PathogenBox_PlateC F10 MMV062221 96 PathogenBox_PlateC F11 MMV688921 72 PathogenBox_PlateC G02 MMV676191 45 PathogenBox_PlateC G03 MMV675993 91 PathogenBox_PlateC G04 MMV021660 89 PathogenBox_PlateC G05 MMV688417 91 PathogenBox_PlateC G06 MMV687273 96 PathogenBox_PlateC G07 MMV687180 76 PathogenBox_PlateC G08 MMV1088520 95 PathogenBox_PlateC G09 MMV688891 28 PathogenBox_PlateC G10 MMV023370 46 PathogenBox_PlateC G11 MMV688703 94 PathogenBox_PlateC H02 MMV675969 80 PathogenBox_PlateC H03 MMV688313 92

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PathogenBox_PlateC H04 MMV687172 92 PathogenBox_PlateC H05 MMV688844 85 PathogenBox_PlateC H06 MMV1198433 40 PathogenBox_PlateC H07 MMV024311 93 PathogenBox_PlateC H08 MMV1019989 52 PathogenBox_PlateC H09 MMV1037162 83 PathogenBox_PlateC H10 MMV689437 93 PathogenBox_PlateC H11 MMV688955 67 PathogenBox_PlateD A02 MMV026468 28 PathogenBox_PlateD A03 MMV020670 90 PathogenBox_PlateD A04 MMV023953 67 PathogenBox_PlateD A05 MMV010576 69 PathogenBox_PlateD A06 MMV032967 76 PathogenBox_PlateD A07 MMV031011 79 PathogenBox_PlateD A08 MMV688178 70 PathogenBox_PlateD A09 MMV688362 93 PathogenBox_PlateD A10 MMV687706 68 PathogenBox_PlateD A11 MMV026356 93 PathogenBox_PlateD B02 MMV011511 92 PathogenBox_PlateD B03 MMV007625 64 PathogenBox_PlateD B04 MMV007471 87 PathogenBox_PlateD B05 MMV024829 88 PathogenBox_PlateD B06 MMV045105 73 PathogenBox_PlateD B07 MMV022029 66 PathogenBox_PlateD B08 MMV676064 87 PathogenBox_PlateD B09 MMV688180 92 PathogenBox_PlateD B10 MMV024035 92 PathogenBox_PlateD B11 MMV688941 82 PathogenBox_PlateD C02 MMV020291 64 PathogenBox_PlateD C03 MMV006833 84 PathogenBox_PlateD C04 MMV026490 80 PathogenBox_PlateD C05 MMV687246 67 PathogenBox_PlateD C06 MMV676162 73 PathogenBox_PlateD C07 MMV024114 67 PathogenBox_PlateD C08 MMV688467 46 PathogenBox_PlateD C09 MMV675998 93 PathogenBox_PlateD C10 MMV659010 86 PathogenBox_PlateD C11 MMV676008 64 PathogenBox_PlateD D02 MMV676269 60 PathogenBox_PlateD D03 MMV020081 71 PathogenBox_PlateD D04 MMV026550 62 PathogenBox_PlateD D05 MMV675995 82 PathogenBox_PlateD D06 MMV688274 71 PathogenBox_PlateD D07 MMV023860 75

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PathogenBox_PlateD D08 MMV688407 93 PathogenBox_PlateD D09 MMV023949 76 PathogenBox_PlateD D10 MMV676050 78 PathogenBox_PlateD D11 MMV024406 46 PathogenBox_PlateD E02 MMV023233 87 PathogenBox_PlateD E03 MMV085230 86 PathogenBox_PlateD E04 MMV085071 93 PathogenBox_PlateD E05 MMV659004 92 PathogenBox_PlateD E06 MMV676260 85 PathogenBox_PlateD E07 MMV688364 71 PathogenBox_PlateD E08 MMV032995 81 PathogenBox_PlateD E09 MMV688279 66 PathogenBox_PlateD E10 MMV688271 93 PathogenBox_PlateD E11 MMV019790 53 PathogenBox_PlateD F02 MMV009135 90 PathogenBox_PlateD F03 MMV011765 91 PathogenBox_PlateD F04 MMV024937 84 PathogenBox_PlateD F05 MMV085499 75 PathogenBox_PlateD F06 MMV023985 88 PathogenBox_PlateD F07 MMV024195 91 PathogenBox_PlateD F08 MMV676063 31 PathogenBox_PlateD F09 MMV676186 77 PathogenBox_PlateD F10 MMV688474 93 PathogenBox_PlateD F11 MMV687812 42 PathogenBox_PlateD G02 MMV007803 89 PathogenBox_PlateD G03 MMV001059 65 PathogenBox_PlateD G04 MMV011691 49 PathogenBox_PlateD G05 MMV676877 78 PathogenBox_PlateD G06 MMV663250 92 PathogenBox_PlateD G07 MMV407539 79 PathogenBox_PlateD G08 MMV688372 84 PathogenBox_PlateD G09 MMV658993 71 PathogenBox_PlateD G10 MMV676182 92 PathogenBox_PlateD G11 MMV676411 93 PathogenBox_PlateD H02 MMV007133 90 PathogenBox_PlateD H03 MMV022478 92 PathogenBox_PlateD H04 MMV024101 92 PathogenBox_PlateD H05 MMV676881 87 PathogenBox_PlateD H06 MMV024443 93 PathogenBox_PlateD H07 MMV688469 84 PathogenBox_PlateD H08 MMV023388 92 PathogenBox_PlateD H09 MMV675968 94 PathogenBox_PlateD H10 MMV675996 84 PathogenBox_PlateD H11 MMV688980 17

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PathogenBox_PlateE A02 MMV011229 93 PathogenBox_PlateE A03 MMV676468 97 PathogenBox_PlateE A04 MMV688771 15 PathogenBox_PlateE A05 MMV687798 77 PathogenBox_PlateE A06 MMV688775 75 PathogenBox_PlateE A07 MMV676159 97 PathogenBox_PlateE A08 MMV393144 11 PathogenBox_PlateE A09 MMV007920 83 PathogenBox_PlateE A10 MMV688270 72 PathogenBox_PlateE A11 MMV019993 84 PathogenBox_PlateE B02 MMV687794 95 PathogenBox_PlateE B03 MMV676470 86 PathogenBox_PlateE B04 MMV688938 97 PathogenBox_PlateE B05 MMV689000 97 PathogenBox_PlateE B06 MMV004168 97 PathogenBox_PlateE B07 MMV676161 78 PathogenBox_PlateE B08 MMV023183 93 PathogenBox_PlateE B09 MMV047015 96 PathogenBox_PlateE B10 MMV688795 96 PathogenBox_PlateE B11 MMV688352 58 PathogenBox_PlateE C02 MMV676398 89 PathogenBox_PlateE C03 MMV676472 89 PathogenBox_PlateE C04 MMV671636 80 PathogenBox_PlateE C05 MMV676599 82 PathogenBox_PlateE C06 MMV689244 97 PathogenBox_PlateE C07 MMV688411 95 PathogenBox_PlateE C08 MMV687765 89 PathogenBox_PlateE C09 MMV020165 57 PathogenBox_PlateE C10 MMV676524 85 PathogenBox_PlateE C11 MMV611037 94 PathogenBox_PlateE D02 MMV688766 9 PathogenBox_PlateE D03 MMV200748 93 PathogenBox_PlateE D04 MMV667494 77 PathogenBox_PlateE D05 MMV028694 91 PathogenBox_PlateE D06 MMV001499 97 PathogenBox_PlateE D07 MMV688345 97 PathogenBox_PlateE D08 MMV010545 89 PathogenBox_PlateE D09 MMV023227 95 PathogenBox_PlateE D10 MMV687700 71 PathogenBox_PlateE D11 MMV676384 94 PathogenBox_PlateE E02 MMV020289 94 PathogenBox_PlateE E03 MMV002816 78 PathogenBox_PlateE E04 MMV634140 85 PathogenBox_PlateE E05 MMV030734 71

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PathogenBox_PlateE E06 MMV689243 97 PathogenBox_PlateE E07 MMV676358 84 PathogenBox_PlateE E08 MMV687729 64 PathogenBox_PlateE E09 MMV407834 64 PathogenBox_PlateE E10 MMV687813 49 PathogenBox_PlateE E11 MMV153413 97 PathogenBox_PlateE F02 MMV019551 96 PathogenBox_PlateE F03 MMV688552 92 PathogenBox_PlateE F04 MMV016838 80 PathogenBox_PlateE F05 MMV676270 86 PathogenBox_PlateE F06 MMV688755 97 PathogenBox_PlateE F07 MMV228911 59 PathogenBox_PlateE F08 MMV272144 97 PathogenBox_PlateE F09 MMV026313 97 PathogenBox_PlateE F10 MMV161996 57 PathogenBox_PlateE F11 MMV688543 81 PathogenBox_PlateE G02 MMV146306 96 PathogenBox_PlateE G03 MMV688557 94 PathogenBox_PlateE G04 MMV021013 97 PathogenBox_PlateE G05 MMV392832 85 PathogenBox_PlateE G06 MMV688754 93 PathogenBox_PlateE G07 MMV001561 97 PathogenBox_PlateE G08 MMV658988 97 PathogenBox_PlateE G09 MMV084864 92 PathogenBox_PlateE G10 MMV676492 97 PathogenBox_PlateE G11 MMV688415 76 PathogenBox_PlateE H02 MMV688330 34 PathogenBox_PlateE H03 MMV687796 27 PathogenBox_PlateE H04 MMV688939 78 PathogenBox_PlateE H05 MMV688978 97 PathogenBox_PlateE H06 MMV688990 83 PathogenBox_PlateE H07 MMV688273 29 PathogenBox_PlateE H08 MMV393995 94 PathogenBox_PlateE H09 MMV1236379 94 PathogenBox_PlateE H10 MMV688550 82 PathogenBox_PlateE H11 MMV495543 84

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7.12 Appendix 10. Profiles and percentage anticrithidial activities of compounds in the GSK T.brucei box.

Plate barcode Position Compound ID Inhibition (%) at 100µM concentration G20SL2B A2 TCMDC-143074 100 G20SL2B G7 TCMDC-143100 46 G20SL2B B8 TCMDC-143121 53 G20SL2B H9 TCMDC-143123 44 G20SL2B A10 TCMDC-143128 53 G20SL2B A7 TCMDC-143131 52 G20SL2B E5 TCMDC-143132 57 G20SL2B C11 TCMDC-143138 91 G20SL2B D11 TCMDC-143154 49 G20SL2B A9 TCMDC-143167 31 G20SL2B E3 TCMDC-143172 84 G20SL2B A3 TCMDC-143176 74 G20SL2B H2 TCMDC-143199 63 G20SL2B H7 TCMDC-143205 55 G20SL2B B11 TCMDC-143206 21 G20SL2B C7 TCMDC-143225 50 G20SL2B E6 TCMDC-143230 40 G20SL2B G9 TCMDC-143233 97 G20SL2B E9 TCMDC-143240 65 G20SL2B B6 TCMDC-143242 82 G20SL2B F10 TCMDC-143251 71 G20SL2B A11 TCMDC-143257 48 G20SL2B F7 TCMDC-143263 100 G20SL2B C9 TCMDC-143264 45 G20SL2B G2 TCMDC-143265 73 G20SL2B E8 TCMDC-143267 78 G20SL2B C4 TCMDC-143270 78 G20SL2B H3 TCMDC-143289 59 G20SL2B H8 TCMDC-143290 32 G20SL2B E2 TCMDC-143292 100 G20SL2B E10 TCMDC-143307 72 G20SL2B G8 TCMDC-143320 63 G20SL2B B7 TCMDC-143323 89 G20SL2B B4 TCMDC-143335 74 G20SL2B B9 TCMDC-143337 59 G20SL2B F11 TCMDC-143342 67 G20SL2B G3 TCMDC-143343 52 G20SL2B A8 TCMDC-143356 88 G20SL2B D7 TCMDC-143359 73

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G20SL2B D8 TCMDC-143361 68 G20SL2B D10 TCMDC-143365 50 G20SL2B D3 TCMDC-143378 74 G20SL2B G5 TCMDC-143399 58 G20SL2B H6 TCMDC-143424 67 G20SL2B D5 TCMDC-143428 100 G20SL2B F9 TCMDC-143444 80 G20SL2B D2 TCMDC-143449 94 G20SL2B C10 TCMDC-143453 59 G20SL2B C8 TCMDC-143454 66 G20SL2B G6 TCMDC-143457 94 G20SL2B B5 TCMDC-143460 100 G20SL2B F3 TCMDC-143462 90 G20SL2B H10 TCMDC-143475 56 G20SL2B G4 TCMDC-143493 60 G20SL2B H5 TCMDC-143505 50 G20SL2B F2 TCMDC-143510 76 G20SL2B H11 TCMDC-143513 40 G20SL2B C2 TCMDC-143515 99 G20SL2B F6 TCMDC-143516 61 G20SL2B A5 TCMDC-143533 97 G20SL2B D9 TCMDC-143551 51 G20SL2B D4 TCMDC-143556 62 G20SL2B F5 TCMDC-143565 76 G20SL2B A6 TCMDC-143572 76 G20SL2B C6 TCMDC-143575 54 G20SL2B E4 TCMDC-143578 72 G20SL2B D6 TCMDC-143579 65 G20SL2B C5 TCMDC-143581 36 G20SL2B B10 TCMDC-143585 69 G20SL2B G11 TCMDC-143587 77 G20SL2B E11 TCMDC-143597 88 G20SL2B A4 TCMDC-143609 98 G20SL2B H4 TCMDC-143624 67 G20SL2B G10 TCMDC-143638 77 G20SL2B F4 TCMDC-143645 59 G20SL2B B3 TCMDC-143080 95 G20SL2B C3 TCMDC-143194 81 G20SL2B B2 TCMDC-143273 68 G20SL2B F8 TCMDC-143497 51 G20SL2B E7 TCMDC-143569 84 G20SL2C H10 TCMDC-142497 94 G20SL2C A2 TCMDC-143073 99 G20SL2C A9 TCMDC-143089 27

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G20SL2C A11 TCMDC-143102 87 G20SL2C E4 TCMDC-143104 89 G20SL2C D8 TCMDC-143107 55 G20SL2C G10 TCMDC-143111 89 G20SL2C G2 TCMDC-143116 70 G20SL2C G9 TCMDC-143125 100 G20SL2C B10 TCMDC-143134 43 G20SL2C D3 TCMDC-143146 68 G20SL2C D2 TCMDC-143158 80 G20SL2C E7 TCMDC-143173 69 G20SL2C B6 TCMDC-143189 45 G20SL2C C8 TCMDC-143195 10 G20SL2C F8 TCMDC-143204 78 G20SL2C F9 TCMDC-143210 80 G20SL2C C10 TCMDC-143219 100 G20SL2C B5 TCMDC-143220 91 G20SL2C A7 TCMDC-143227 10 G20SL2C A3 TCMDC-143228 16 G20SL2C C6 TCMDC-143229 15 G20SL2C C9 TCMDC-143231 58 G20SL2C B9 TCMDC-143243 100 G20SL2C A10 TCMDC-143250 85 G20SL2C G4 TCMDC-143283 30 G20SL2C E10 TCMDC-143294 49 G20SL2C G7 TCMDC-143303 15 G20SL2C F3 TCMDC-143326 67 G20SL2C H2 TCMDC-143339 87 G20SL2C D11 TCMDC-143341 46 G20SL2C H3 TCMDC-143352 77 G20SL2C C3 TCMDC-143357 56 G20SL2C C5 TCMDC-143360 100 G20SL2C D4 TCMDC-143363 100 G20SL2C H11 TCMDC-143366 76 G20SL2C H5 TCMDC-143368 23 G20SL2C G5 TCMDC-143369 74 G20SL2C A8 TCMDC-143370 90 G20SL2C G8 TCMDC-143373 19 G20SL2C C11 TCMDC-143374 88 G20SL2C G11 TCMDC-143380 83 G20SL2C E11 TCMDC-143386 80 G20SL2C H6 TCMDC-143390 44 G20SL2C D6 TCMDC-143393 65 G20SL2C E2 TCMDC-143394 84 G20SL2C D7 TCMDC-143400 28

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G20SL2C B11 TCMDC-143401 90 G20SL2C H4 TCMDC-143402 90 G20SL2C F11 TCMDC-143412 12 G20SL2C H7 TCMDC-143425 86 G20SL2C F6 TCMDC-143435 87 G20SL2C A6 TCMDC-143436 50 G20SL2C E5 TCMDC-143438 50 G20SL2C F4 TCMDC-143445 68 G20SL2C E9 TCMDC-143446 79 G20SL2C D10 TCMDC-143452 25 G20SL2C G6 TCMDC-143456 62 G20SL2C C4 TCMDC-143458 66 G20SL2C B7 TCMDC-143496 61 G20SL2C D9 TCMDC-143499 56 G20SL2C E3 TCMDC-143525 72 G20SL2C E6 TCMDC-143540 58 G20SL2C E8 TCMDC-143543 88 G20SL2C B4 TCMDC-143547 81 G20SL2C A4 TCMDC-143580 12 G20SL2C F2 TCMDC-143582 88 G20SL2C C7 TCMDC-143589 63 G20SL2C D5 TCMDC-143595 80 G20SL2C G3 TCMDC-143596 82 G20SL2C B8 TCMDC-143619 83 G20SL2C H8 TCMDC-143634 12 G20SL2C C2 TCMDC-143640 58 G20SL2C F5 TCMDC-143641 93 G20SL2C B2 TCMDC-143642 76 G20SL2C B3 TCMDC-143643 80 G20SL2C F7 TCMDC-143644 72 G20SL2C F10 TCMDC-143648 57 G20SL2C A5 TCMDC-143130 65 G20SL2C H9 TCMDC-143526 94 G211HCO D2 TCMDC-143226 14 G211HCO C2 TCMDC-143316 99 G211HCO F2 TCMDC-143330 20 G211HCO G2 TCMDC-143377 73 G211HCO A3 TCMDC-143382 70 G211HCO H2 TCMDC-143392 92 G211HCO B2 TCMDC-143468 82 G211HCO E2 TCMDC-143646 91 G211HCO A2 TCMDC-143163 29 G211IMU E2 TCMDC-142716 81 G211IMU G2 TCMDC-143079 93

115

G211IMU A2 TCMDC-143112 100 G211IMU C2 TCMDC-143192 61 G211IMU H2 TCMDC-143254 60 G211IMU A3 TCMDC-143299 92 G211IMU B2 TCMDC-143318 50 G211IMU D2 TCMDC-143364 46 G211IMU C3 TCMDC-143466 54 G211IMU F2 TCMDC-143469 92 G211IMU D3 TCMDC-143471 90 G211IMU B3 TCMDC-143636 58 G211IRO B2 TCMDC-143312 35 G211IRO C2 TCMDC-143470 72 G211IRO E2 TCMDC-143544 51 G211IRO A2 TCMDC-143583 36 G211IRO D2 TCMDC-143635 48 G214GU7 B2 TCMDC-143312 28 G214GU7 A2 TCMDC-143583 58

7.13 Appendix 11. Profiles and percentage anticrithidial activities of compounds in the GSK T. cruzi box.

Plate barcode Position Compound ID Inhibition (%) at 100µM concentration G20SL2D H9 TCMDC-143081 70 G20SL2D E11 TCMDC-143083 95 G20SL2D E2 TCMDC-143088 100 G20SL2D G5 TCMDC-143097 21 G20SL2D C8 TCMDC-143108 55 G20SL2D H4 TCMDC-143114 87 G20SL2D G10 TCMDC-143135 91 G20SL2D A5 TCMDC-143142 100 G20SL2D G2 TCMDC-143143 57 G20SL2D A6 TCMDC-143148 14 G20SL2D A4 TCMDC-143149 100 G20SL2D H3 TCMDC-143150 87 G20SL2D A10 TCMDC-143152 100 G20SL2D B4 TCMDC-143155 96 G20SL2D G11 TCMDC-143156 68 G20SL2D F2 TCMDC-143157 13 G20SL2D H5 TCMDC-143161 67 G20SL2D B2 TCMDC-143162 93 G20SL2D E3 TCMDC-143178 74

116

G20SL2D A7 TCMDC-143187 37 G20SL2D F3 TCMDC-143190 67 G20SL2D C10 TCMDC-143200 67 G20SL2D B10 TCMDC-143203 97 G20SL2D E10 TCMDC-143207 96 G20SL2D C3 TCMDC-143222 99 G20SL2D H10 TCMDC-143224 70 G20SL2D E6 TCMDC-143235 100 G20SL2D F10 TCMDC-143241 10 G20SL2D B5 TCMDC-143244 76 G20SL2D E4 TCMDC-143247 89 G20SL2D A11 TCMDC-143248 46 G20SL2D B6 TCMDC-143253 94 G20SL2D A9 TCMDC-143256 54 G20SL2D D3 TCMDC-143258 26 G20SL2D F9 TCMDC-143262 100 G20SL2D G7 TCMDC-143272 94 G20SL2D B9 TCMDC-143276 96 G20SL2D D10 TCMDC-143279 96 G20SL2D C11 TCMDC-143284 65 G20SL2D B7 TCMDC-143300 68 G20SL2D A2 TCMDC-143309 76 G20SL2D H8 TCMDC-143313 14 G20SL2D F7 TCMDC-143319 20 G20SL2D H11 TCMDC-143324 84 G20SL2D D9 TCMDC-143328 73 G20SL2D B8 TCMDC-143329 100 G20SL2D G4 TCMDC-143332 55 G20SL2D D6 TCMDC-143346 68 G20SL2D C2 TCMDC-143362 42 G20SL2D D2 TCMDC-143384 26 G20SL2D G6 TCMDC-143387 81 G20SL2D E8 TCMDC-143405 80 G20SL2D H7 TCMDC-143408 65 G20SL2D D7 TCMDC-143409 95 G20SL2D D8 TCMDC-143411 68 G20SL2D C4 TCMDC-143415 100 G20SL2D E7 TCMDC-143421 90 G20SL2D G3 TCMDC-143434 18 G20SL2D C5 TCMDC-143439 90 G20SL2D D11 TCMDC-143461 100 G20SL2D A3 TCMDC-143504 66 G20SL2D D4 TCMDC-143507 47 G20SL2D B11 TCMDC-143527 33

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G20SL2D F8 TCMDC-143529 100 G20SL2D A8 TCMDC-143535 55 G20SL2D D5 TCMDC-143537 65 G20SL2D F6 TCMDC-143541 100 G20SL2D E9 TCMDC-143552 69 G20SL2D F11 TCMDC-143555 93 G20SL2D G9 TCMDC-143561 41 G20SL2D C9 TCMDC-143598 54 G20SL2D E5 TCMDC-143602 100 G20SL2D G8 TCMDC-143605 8 G20SL2D C7 TCMDC-143608 100 G20SL2D H6 TCMDC-143611 84 G20SL2D H2 TCMDC-143616 18 G20SL2D B3 TCMDC-143617 35 G20SL2D C6 TCMDC-143620 86 G20SL2D F4 TCMDC-143632 87 G20SL2D F5 TCMDC-143637 42 G211GWY B2 TCMDC-143080 89 G211GWY C5 TCMDC-143087 100 G211GWY A7 TCMDC-143109 100 G211GWY E2 TCMDC-143130 74 G211GWY G5 TCMDC-143137 70 G211GWY G3 TCMDC-143160 95 G211GWY A3 TCMDC-143163 52 G211GWY B3 TCMDC-143164 69 G211GWY E6 TCMDC-143186 99 G211GWY A6 TCMDC-143193 71 G211GWY C2 TCMDC-143194 81 G211GWY C3 TCMDC-143197 80 G211GWY A2 TCMDC-143273 57 G211GWY A4 TCMDC-143308 100 G211GWY D3 TCMDC-143315 100 G211GWY D6 TCMDC-143325 100 G211GWY F4 TCMDC-143331 97 G211GWY F6 TCMDC-143371 94 G211GWY F3 TCMDC-143414 74 G211GWY C4 TCMDC-143430 100 G211GWY F2 TCMDC-143446 67 G211GWY C6 TCMDC-143455 100 G211GWY D4 TCMDC-143464 100 G211GWY G4 TCMDC-143477 100 G211GWY E4 TCMDC-143481 100 G211GWY A5 TCMDC-143492 100 G211GWY B6 TCMDC-143494 100

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G211GWY D2 TCMDC-143497 85 G211GWY H5 TCMDC-143498 100 G211GWY G6 TCMDC-143506 100 G211GWY G2 TCMDC-143526 75 G211GWY E3 TCMDC-143539 100 G211GWY D5 TCMDC-143542 100 G211GWY H6 TCMDC-143550 100 G211GWY E5 TCMDC-143562 100 G211GWY H2 TCMDC-143569 63 G211GWY H4 TCMDC-143590 100 G211GWY B5 TCMDC-143593 100 G211GWY H3 TCMDC-143599 96 G211GWY B4 TCMDC-143601 100 G211GWY F5 TCMDC-143614 100 G211GWZ B2 TCMDC-123621 100 G211GWZ G2 TCMDC-125222 100 G211GWZ F2 TCMDC-139489 22 G211GWZ E2 TCMDC-140766 33 G211GWZ A3 TCMDC-143071 53 G211GWZ F8 TCMDC-143082 68 G211GWZ C9 TCMDC-143084 61 G211GWZ A10 TCMDC-143103 22 G211GWZ E6 TCMDC-143105 56 G211GWZ E9 TCMDC-143120 71 G211GWZ A9 TCMDC-143126 96 G211GWZ C2 TCMDC-143127 100 G211GWZ B10 TCMDC-143151 100 G211GWZ G6 TCMDC-143153 44 G211GWZ G9 TCMDC-143159 100 G211GWZ C11 TCMDC-143179 18 G211GWZ C6 TCMDC-143182 71 G211GWZ H10 TCMDC-143191 62 G211GWZ C3 TCMDC-143209 22 G211GWZ H5 TCMDC-143232 67 G211GWZ H8 TCMDC-143275 70 G211GWZ G8 TCMDC-143282 57 G211GWZ A4 TCMDC-143286 100 G211GWZ F7 TCMDC-143288 39 G211GWZ B3 TCMDC-143291 32 G211GWZ D2 TCMDC-143298 13 G211GWZ A6 TCMDC-143301 69 G211GWZ H4 TCMDC-143302 56 G211GWZ A11 TCMDC-143304 100 G211GWZ E3 TCMDC-143310 100

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G211GWZ B5 TCMDC-143311 33 G211GWZ A2 TCMDC-143314 10 G211GWZ D7 TCMDC-143317 45 G211GWZ F10 TCMDC-143333 59 G211GWZ B6 TCMDC-143334 60 G211GWZ D3 TCMDC-143336 48 G211GWZ B11 TCMDC-143338 17 G211GWZ G11 TCMDC-143354 71 G211GWZ C10 TCMDC-143372 76 G211GWZ H11 TCMDC-143389 82 G211GWZ E7 TCMDC-143403 44 G211GWZ D4 TCMDC-143410 79 G211GWZ G7 TCMDC-143416 100 G211GWZ H2 TCMDC-143417 35 G211GWZ F4 TCMDC-143422 100 G211GWZ D6 TCMDC-143423 67 G211GWZ F11 TCMDC-143426 85 G211GWZ F3 TCMDC-143432 100 G211GWZ D8 TCMDC-143437 88 G211GWZ E8 TCMDC-143440 100 G211GWZ A5 TCMDC-143463 69 G211GWZ H9 TCMDC-143465 50 G211GWZ H6 TCMDC-143467 95 G211GWZ C7 TCMDC-143474 74 G211GWZ E4 TCMDC-143476 79 G211GWZ G5 TCMDC-143479 73 G211GWZ F5 TCMDC-143484 45 G211GWZ E5 TCMDC-143490 79 G211GWZ A8 TCMDC-143495 100 G211GWZ C4 TCMDC-143502 100 G211GWZ B4 TCMDC-143511 100 G211GWZ G10 TCMDC-143519 56 G211GWZ A7 TCMDC-143520 95 G211GWZ G3 TCMDC-143528 44 G211GWZ D11 TCMDC-143530 67 G211GWZ F9 TCMDC-143545 51 G211GWZ C8 TCMDC-143546 61 G211GWZ H7 TCMDC-143548 48 G211GWZ E11 TCMDC-143549 93 G211GWZ F6 TCMDC-143553 89 G211GWZ E10 TCMDC-143559 76 G211GWZ B9 TCMDC-143564 38 G211GWZ C5 TCMDC-143588 52 G211GWZ D10 TCMDC-143592 88

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G211GWZ D5 TCMDC-143604 91 G211GWZ D9 TCMDC-143613 58 G211GWZ B7 TCMDC-143615 14 G211GWZ H3 TCMDC-143623 81 G211GWZ B8 TCMDC-143626 25 G211GWZ G4 TCMDC-143631 73 G211IMQ D2 TCMDC-143221 71 G211IMQ C3 TCMDC-143293 89 G211IMQ G2 TCMDC-143376 75 G211IMQ B3 TCMDC-143379 90 G211IMQ C2 TCMDC-143381 60 G211IMQ B2 TCMDC-143385 60 G211IMQ E3 TCMDC-143413 66 G211IMQ E2 TCMDC-143433 57 G211IMQ F3 TCMDC-143466 60 G211IMQ H2 TCMDC-143606 98 G211IMQ F2 TCMDC-143610 95 G211IMQ D3 TCMDC-143612 100 G211IMQ A3 TCMDC-143622 100 G211IMQ A2 TCMDC-143625 100 Q203FAM C2 TCMDC-143085 47 Q203FAM A2 TCMDC-143118 44 Q203FAM B2 TCMDC-143185 86 Q203FAM D2 TCMDC-143500 77

7.14 Appendix 12. Profiles and percentage anticrithidial activities of compounds in the GSK Leishmania box

Plate barcode Position Compound ID Inhibition (%) at 100µM concentration G211IMR B8 TCMDC-125387 83 G211IMR C8 TCMDC-142900 100 G211IMR B7 TCMDC-143075 96 G211IMR F7 TCMDC-143098 82 G211IMR E5 TCMDC-143099 82 G211IMR F6 TCMDC-143101 84 G211IMR B2 TCMDC-143129 100 G211IMR E8 TCMDC-143133 75 G211IMR E4 TCMDC-143139 81

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G211IMR D5 TCMDC-143140 70 G211IMR C5 TCMDC-143144 84 G211IMR H6 TCMDC-143170 95 G211IMR H3 TCMDC-143171 81 G211IMR C4 TCMDC-143184 91 G211IMR A7 TCMDC-143188 100 G211IMR C2 TCMDC-143201 95 G211IMR D3 TCMDC-143202 100 G211IMR F8 TCMDC-143218 100 G211IMR G7 TCMDC-143236 92 G211IMR A8 TCMDC-143237 98 G211IMR E2 TCMDC-143245 85 G211IMR F4 TCMDC-143246 80 G211IMR G2 TCMDC-143252 98 G211IMR E3 TCMDC-143266 92 G211IMR B6 TCMDC-143268 100 G211IMR H4 TCMDC-143278 100 G211IMR D7 TCMDC-143287 92 G211IMR E7 TCMDC-143306 100 G211IMR B4 TCMDC-143340 84 G211IMR C7 TCMDC-143345 68 G211IMR G6 TCMDC-143347 72 G211IMR A4 TCMDC-143353 74 G211IMR H7 TCMDC-143375 100 G211IMR D8 TCMDC-143404 94 G211IMR E6 TCMDC-143407 82 G211IMR C3 TCMDC-143418 77 G211IMR A5 TCMDC-143419 100 G211IMR F2 TCMDC-143427 90 G211IMR F3 TCMDC-143431 94 G211IMR G4 TCMDC-143443 100 G211IMR A6 TCMDC-143491 78 G211IMR G5 TCMDC-143508 100 G211IMR B3 TCMDC-143514 81 G211IMR F5 TCMDC-143523 73 G211IMR G3 TCMDC-143536 67 G211IMR A2 TCMDC-143538 79 G211IMR H2 TCMDC-143557 87 G211IMR H5 TCMDC-143558 80 G211IMR A3 TCMDC-143570 90 G211IMR D2 TCMDC-143573 97 G211IMR D4 TCMDC-143574 100 G211IMR H8 TCMDC-143576 95 G211IMR B5 TCMDC-143577 75

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G211IMR C6 TCMDC-143591 92 G211IMR G8 TCMDC-143600 100 G211IMR B9 TCMDC-125387 69 G211IMS D4 TCMDC-143163 66 G211IMS A9 TCMDC-143164 75 G211IMS C5 TCMDC-143197 77 G211IMS H11 TCMDC-143315 98 G211IMS D2 TCMDC-125160 89 G211IMS G5 TCMDC-125826 100 G211IMS A5 TCMDC-143086 57 G211IMS A8 TCMDC-143094 100 G211IMS G7 TCMDC-143106 96 G211IMS B9 TCMDC-143110 99 G211IMS F2 TCMDC-143113 100 G211IMS E3 TCMDC-143115 98 G211IMS B5 TCMDC-143119 71 G211IMS H10 TCMDC-143124 72 G211IMS B4 TCMDC-143136 100 G211IMS C6 TCMDC-143141 76 G211IMS H9 TCMDC-143145 100 G211IMS A3 TCMDC-143147 96 G211IMS F8 TCMDC-143165 100 G211IMS F7 TCMDC-143166 100 G211IMS E8 TCMDC-143168 76 G211IMS B8 TCMDC-143169 73 G211IMS A6 TCMDC-143174 97 G211IMS G8 TCMDC-143175 93 G211IMS H5 TCMDC-143181 56 G211IMS A10 TCMDC-143196 82 G211IMS D8 TCMDC-143208 91 G211IMS D7 TCMDC-143214 74 G211IMS A11 TCMDC-143223 92 G211IMS D3 TCMDC-143239 100 G211IMS C4 TCMDC-143249 74 G211IMS B6 TCMDC-143255 94 G211IMS D9 TCMDC-143259 78 G211IMS D6 TCMDC-143261 78 G211IMS D11 TCMDC-143269 59 G211IMS F6 TCMDC-143271 100 G211IMS D5 TCMDC-143274 100 G211IMS F10 TCMDC-143277 99 G211IMS H2 TCMDC-143280 93 G211IMS C7 TCMDC-143281 99 G211IMS G2 TCMDC-143295 92

123

G211IMS C8 TCMDC-143297 100 G211IMS F5 TCMDC-143305 85 G211IMS G3 TCMDC-143327 65 G211IMS C10 TCMDC-143344 75 G211IMS E11 TCMDC-143351 72 G211IMS B2 TCMDC-143355 74 G211IMS H7 TCMDC-143358 95 G211IMS G4 TCMDC-143367 74 G211IMS H8 TCMDC-143383 76 G211IMS C9 TCMDC-143388 71 G211IMS B10 TCMDC-143391 71 G211IMS E10 TCMDC-143396 63 G211IMS H6 TCMDC-143398 97 G211IMS E2 TCMDC-143406 96 G211IMS C3 TCMDC-143442 100 G211IMS E5 TCMDC-143447 100 G211IMS H4 TCMDC-143451 75 G211IMS A2 TCMDC-143473 78 G211IMS F4 TCMDC-143483 74 G211IMS G6 TCMDC-143501 88 G211IMS G9 TCMDC-143509 100 G211IMS B11 TCMDC-143518 96 G211IMS C11 TCMDC-143522 78 G211IMS A7 TCMDC-143524 84 G211IMS F3 TCMDC-143531 63 G211IMS G10 TCMDC-143534 91 G211IMS G11 TCMDC-143554 100 G211IMS H3 TCMDC-143563 66 G211IMS C2 TCMDC-143566 72 G211IMS E7 TCMDC-143567 91 G211IMS E9 TCMDC-143571 100 G211IMS F9 TCMDC-143584 78 G211IMS B7 TCMDC-143586 100 G211IMS A4 TCMDC-143603 100 G211IMS E4 TCMDC-143607 99 G211IMS B3 TCMDC-143618 66 G211IMS D10 TCMDC-143621 94 G211IMS F11 TCMDC-143628 72 G211IMS E6 TCMDC-143629 90 G211IMT G4 TCMDC-124508 60 G211IMT C3 TCMDC-142704 100 G211IMT B3 TCMDC-143072 82 G211IMT C5 TCMDC-143076 70 G211IMT B5 TCMDC-143077 96

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G211IMT E4 TCMDC-143078 80 G211IMT C2 TCMDC-143117 65 G211IMT A5 TCMDC-143211 79 G211IMT A4 TCMDC-143212 100 G211IMT C4 TCMDC-143213 82 G211IMT D4 TCMDC-143215 77 G211IMT B4 TCMDC-143216 96 G211IMT D5 TCMDC-143217 89 G211IMT H4 TCMDC-143285 100 G211IMT A3 TCMDC-143296 96 G211IMT H3 TCMDC-143350 66 G211IMT E2 TCMDC-143397 100 G211IMT F3 TCMDC-143429 66 G211IMT G2 TCMDC-143448 94 G211IMT D3 TCMDC-143478 74 G211IMT D2 TCMDC-143480 66 G211IMT H2 TCMDC-143482 70 G211IMT E5 TCMDC-143488 100 G211IMT F2 TCMDC-143517 100 G211IMT F5 TCMDC-143521 90 G211IMT A2 TCMDC-143532 74 G211IMT F4 TCMDC-143568 100 G211IMT B2 TCMDC-143594 81 G211IMT G3 TCMDC-143633 78 G211IMT E3 TCMDC-143647 100 Q203FAN A4 TCMDC-134026 97 Q203FAN D2 TCMDC-143090 98 Q203FAN H2 TCMDC-143091 95 Q203FAN A3 TCMDC-143092 93 Q203FAN A2 TCMDC-143093 98 Q203FAN B3 TCMDC-143095 91 Q203FAN E2 TCMDC-143096 98 Q203FAN F2 TCMDC-143238 97 Q203FAN D3 TCMDC-143260 75 Q203FAN G2 TCMDC-143348 90 Q203FAN C3 TCMDC-143349 75 Q203FAN G3 TCMDC-143459 69 Q203FAN B2 TCMDC-143486 80 Q203FAN F3 TCMDC-143487 97 Q203FAN C2 TCMDC-143503 100 Q203FAN H3 TCMDC-143512 66 Q203FAN E3 TCMDC-143630 83

125